Structured substrates for improving detection of light emissions and methods relating to the same

ABSTRACT

A structured substrate includes a substrate body having an active side. The substrate body includes reaction cavities that open along the active side and interstitial regions that separate the reaction cavities. The structured substrate includes an ensemble amplifier positioned within each of the reaction cavities. The ensemble amplifier includes a plurality of nanostructures configured to at least one of amplify electromagnetic energy that propagates into the corresponding reaction cavity or amplify electromagnetic energy that is generated within the corresponding reaction cavity.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/564,174, filed on Oct. 3, 2017, which is a national stageentry of International Patent Application No. PCT/US2016/027399, filedon Apr. 14, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/147,440, filed on Apr. 14, 2015, all of which areincorporated herein by reference in their entirety.

BACKGROUND

The present disclosure relates generally to biological or chemicalanalysis and more particularly to systems and methods for detectinglight emissions from an array of reaction sites.

Various protocols in biological or chemical research involve performinga large number of controlled reactions at localized areas of a supportsurface or within reaction cavities. The designated reactions may thenbe observed or detected and subsequent analysis may help identify orreveal properties of chemicals involved in the reaction. For example, insome multiplex assays, an unknown analyte having an identifiable label(e.g., fluorescent label) may be exposed to thousands of known probesunder controlled conditions. Each known probe may be deposited into acorresponding well of a microplate. Observing any chemical reactionsthat occur between the known probes and the unknown analyte within thewells may help identify or reveal properties of the analyte.

Other protocols that detect light emissions from an array of reactionsites include known DNA sequencing protocols, such assequencing-by-synthesis (SBS) or cyclic-array sequencing. In SBS, aplurality of fluorescently-labeled nucleotides are used to sequencenucleic acids of numerous clusters (or clonal populations) of amplifiedDNA that are located on the surface of a substrate. The surface may, forexample, define a channel in a flow cell. The sequences of the nucleicacids in the different clusters are determined by running numerouscycles in which a fluorescently-labeled nucleotide is added to thecluster and then excited by a light source to provide light emissions.

Although the sequencing systems currently used are effective inidentifying the nucleotides and determining a sequence of the nucleicacids, systems that are more cost-effective and/or that achieve an evensmaller error rate are desired. For example, it is desirable to increasethe density of reaction sites. Sequencing methodologies andcorresponding systems, however, exploit a complex collection oftechnologies. Improvements in some of these technologies have been shownto provide substantial cost reductions. However, it is difficult topredict which, if any, is amenable to cost-reducing improvements. Giventhe dependencies between the technologies in the sequencing systems itis even more difficult to predict which can be modified without havingan adverse impact on the overall performance of the methodology orsystem.

One challenge confronted by many protocols is detecting, with a suitablelevel of confidence, the designated reactions that generate lightemissions. This challenge is even more difficult as the reaction sitesbecome smaller and the density of reaction sites becomes greater. Forexample, reaction sites may have a diameter or width that is 750 nm orless, and adjacent reaction sites may be separated by 750 nm or less.One consequence of the reaction sites becoming smaller is that theamount of generated light emissions also becomes smaller and,consequently, more challenging to detect. Moreover, as the density ofreaction sites becomes greater, it may be more difficult to distinguishwhich reaction sites provided the light emissions. In addition to theabove, it is generally desirable to decrease the amount of time used fordetecting the light emissions (also referred to as scan time or imagetime). As scan times decrease, fewer photons are detected, therebyrendering it even more challenging to reliably detect light emissionsthat are indicative of a designated reaction occurring.

Accordingly, a need exists for apparatuses, systems, and methods thatgenerate a sufficient amount of light for detecting designated reactionswithin an array of reaction sites.

BRIEF SUMMARY

Presented herein are structures substrates and methods for manufacturingstructures substrates that improve the detectability of opticalemissions provided by discrete reaction sites. For example, thestructures substrates may increase an intensity of an excitation lightexperienced by biological substances at the discrete sites, may increasean intensity of the optical emissions from the biological substances,and/or may control a directionality of the optical emissions. Alsopresented herein are methods of detecting optical emissions from anarray of discrete sites. The discrete sites may be reaction cavitiesformed within a substrate body or localized areas along a surface of adevice substrate. The optical emissions may be generated by, forexample, fluorescence, chemiluminescence, bioluminescence,electroluminescence, radioluminescence, and the like. Also presentedherein are structured substrates having a greater density of discretesites (or smaller pitch between adjacent sites) than known systems andmethods of manufacturing the same.

In some embodiments, the methods and structured substrates may beconfigured to enhance the light emissions of fluorescently-labeledsamples and, more specifically, fluorescently-labeled nucleic acids. Inparticular embodiments, the methods and compositions presented hereinprovide fluorescent enhancement of DNA clusters in sequencing bysynthesis reactions involving dye-labeled nucleotides. However, itshould be understood that methods and devices described herein may alsobe suitable for other applications.

In an embodiment, a structured substrate is provided. The structuredsubstrate includes a substrate body having an active side. The substratebody includes reaction cavities that open along the active side andinterstitial regions that separate the reaction cavities. The structuredsubstrate includes an ensemble amplifier positioned within each of thereaction cavities. The ensemble amplifier includes a plurality ofnanostructures configured to at least one of amplify electromagneticenergy that propagates into the corresponding reaction cavity or amplifyelectromagnetic energy that is generated within the correspondingreaction cavity.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base sideand forming nanostructures along the base side of the base layer. Themethod also includes forming a cavity layer that is stacked above thebase side. The cavity layer includes a plurality of reaction cavities inwhich each reaction cavity includes a plurality of the nanostructurestherein. The plurality of nanostructures form an ensemble amplifier ofthe corresponding reaction cavity that is configured to at least one ofamplify electromagnetic energy propagating into the correspondingreaction cavity or amplify electromagnetic energy generated within thecorresponding reaction cavity

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base sideand forming nanostructures along the base side of the base layer. Themethod also includes providing a nanoimprint lithography (NIL) layerover the array of nanostructures. The method also includes imprinting anarray of reaction cavities into the NIL layer, wherein a differentsub-array of the nanostructures is positioned under each reactioncavity. Each sub-array of nanostructures is surrounded by a respectivefill region of the NIL layer. The method also includes removing therespective fill regions of the NIL layer to expose the sub-arrays ofnanostructures within the corresponding reactions cavities. Thesub-array of nanostructures within each reaction cavity forming anensemble amplifier of the corresponding reaction cavity that isconfigured to at least one of amplify electromagnetic energy propagatinginto the corresponding reaction cavity or amplify electromagnetic energygenerated within the corresponding reaction cavity.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base sideand providing a nanoimprint lithography (NIL) layer along the base side.The method also includes imprinting the NIL layer to form a base portionand an array of nanobodies that project from the base portion. Themethod also includes depositing a plasmon resonant film that covers thenanobodies to form a plurality of nanostructures. Each nanostructureincludes a corresponding nanobody and a portion of the plasmon resonantfilm. The method also includes forming a cavity layer including aplurality of reaction cavities in which each reaction cavity includes aplurality of the nanostructures therein. The plurality of nanostructuresform an ensemble amplifier of the corresponding reaction cavity that isconfigured to at least one of amplify electromagnetic energy propagatinginto the corresponding reaction cavity or amplify electromagnetic energygenerated within the corresponding reaction cavity.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having aside surface and an array of reaction cavities. Each of the reactioncavities has an opening along the side surface and extending a depthfrom the corresponding opening into the working substrate. The reactioncavities coincide with an array plane. The method also includesdirecting a deposition stream onto the working substrate at anon-orthogonal angle with respect to the array plane. The depositionstream includes a plasmon resonant material. The working substrate formsa shadow area and an incident area in each reaction cavity relative to apath of the deposition stream such that the plasmon resonant material ofthe deposition stream is blocked by the side surface from beingdeposited onto the shadow area and is permitted to pass through theopening and form along the incident area.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes (a) providing a working substrate having aside surface and an array of reaction cavities. Each of the reactioncavities has an opening along the side surface and extending a depthfrom the corresponding opening into the working substrate, the reactioncavities coinciding with an array plane. The method also includes (b)positioning the working substrate in a receiving orientation relative toa material source. The method also includes (c) directing a depositionstream from the material source onto the working substrate at anon-orthogonal angle with respect to the array plane. The depositionstream includes a plasmon resonant material. The working substrate formsa shadow area and an incident area in each reaction cavity when in thereceiving orientation such that the plasmon resonant material from thedeposition stream is blocked by the side surface from being depositedonto the shadow area and is permitted to pass through the opening andform along the incident area.

Accordingly, one embodiment presented herein is a substrate, comprising:a plurality of nanostructures distributed on a solid support; a gelmaterial forming a layer in association with the plurality ofnanostructures; and a library of target nucleic acids in the gelmaterial. In certain embodiments, the nanostructures are formed of aplasmon resonant material. In certain embodiments, the plasmon resonantmaterial comprises a material selected from the group consisting of:Gold (Au), Silver (Ag), Tin (Sn) Rhodium (Rh), Ruthenium (Ru), Palladium(Pd), Osmium (Os), Iridium (Ir), Platinum (Pt), Titanium (Ti) andAluminum (Al), Chromium (Cr), Copper (Cu), p-type doped silicon, n-typedoped silicon, and gallium arsenide. The plasmon resonant material maycomprise a metallic alloy. For example, the plasmon resonant materialmay comprise Zinc-Indium-Tin Oxide (ZITO) or Tantalum Oxide (e.g.,TaO₅). In certain embodiments, gel material covers the nanostructures.In certain embodiments, the solid support comprises a surface of a flowcell. In certain embodiments, the solid support comprises a planarsurface having a plurality of wells, the nanostructures beingdistributed within the plurality of wells.

Also presented herein is a method of making a substrate, comprising: (a)providing a solid support comprising a planar surface; (b) dispersing aplurality of nanostructures on the surface of the solid support; (c) andcoating at least a portion of the solid support with a gel materialthereby forming a gel layer covering the plurality of nanostructures. Incertain embodiments, the nanostructures are formed of a plasmon resonantmaterial. In certain embodiments of this method, steps (b) and (c) areperformed simultaneously. In certain embodiments, step (b) is performedprior to step (c). In certain embodiments, the method can furthercomprise (d) delivering a library of target nucleic acids to the gelmaterial to produce an array of nucleic acid features in the gelmaterial. In some embodiments, each feature comprises a differentnucleic acid species. In certain embodiments, the plasmon resonantmaterial comprises a material selected from the group consisting of:Gold (Au), Silver (Ag), Tin (Sn) Rhodium (Rh), Ruthenium (Ru), Palladium(Pd), Osmium (Os), Iridium (Ir), Platinum (Pt), Titanium (Ti) andAluminum (Al), Chromium (Cr), Copper (Cu), p-type doped silicon, n-typedoped silicon, and gallium arsenide. The plasmon resonant material maycomprise a metallic alloy. For example, the plasmon resonant materialmay comprise Zinc-Indium-Tin Oxide (ZITO) or Tantalum Oxide (e.g.,TaO₅).

Also presented herein is a method of detecting nucleic acids,comprising: providing a solid support comprising a plurality ofnanostructures; a gel material forming a layer covering the plurality ofnanostructures; and a library of target nucleic acids in the gelmaterial; contacting the solid support with at least one fluorescentlylabeled probe that binds to the target nucleic acids; and detectingfluorescent signal on the solid support to distinguish the targetnucleic acids that bind to the at least one probe. In certainembodiments, the nanostructures are formed of a plasmon resonantmaterial. In certain embodiments, the plasmon resonant materialcomprises a material selected from the group consisting of: Gold (Au),Silver (Ag), Tin (Sn) Rhodium (Rh), Ruthenium (Ru), Palladium (Pd),Osmium (Os), Iridium (Ir), Platinum (Pt), Titanium (Ti) and Aluminum(Al), Chromium (Cr), Copper (Cu), p-type doped silicon, n-type dopedsilicon, and gallium arsenide. The plasmon resonant material maycomprise a metallic alloy. For example, the plasmon resonant materialmay comprise Zinc-Indium-Tin Oxide (ZITO) or Tantalum Oxide (e.g.,TaO₅). In certain embodiments, the solid support comprises a surface ofa flow cell. In certain embodiments, the solid support comprises planarsurface having a plurality of wells, the nanostructures distributedamong the plurality of wells. In certain embodiments, the fluorescentlylabeled probe comprises a fluorescently labeled nucleotide. In certainembodiments, the fluorescently labeled probe comprises a fluorescentlylabeled oligonucleotide. In certain embodiments, detecting comprisesdetection of hybridization of an oligonucleotide probe to target nucleicacids in each feature. In certain embodiments, detecting comprisesdetection of incorporation of a nucleotide or an oligonucleotide probeto target nucleic acids in each feature.

Also presented herein is an array, comprising: a solid supportcomprising a surface, the surface comprising a plurality of wells, thewells being separated from each other by interstitial regions; and aplurality of nanostructures in each of said plurality of wells. Incertain embodiments, the nanostructures are plasmonic nanostructures. Incertain embodiments, the nanostructures are situated at the bottom ofthe wells. In certain embodiments, the nanostructures are situated alongthe walls of the wells. In certain embodiments, the interstitial regionsare substantially devoid of nanostructures. In certain embodiments, thenanostructures comprise nanostructures. In certain embodiments, thenanostructures have a diameter of greater than 1 nm, 2 nm, 3 nm, 4 nm, 5nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70nm, 80 nm, 90 nm or greater than 100 nm. In certain embodiments, thenanostructures have a diameter of less than 100 nm, 90 nm, 80 nm, 70 nm,60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm,4 nm, 3 nm, 2 nm, or less than 1 nm. In certain embodiments, thenanostructures comprise dimers or trimers within the wells. In certainembodiments, the nanostructures comprise bowtie nanoantennae. In certainembodiments, the nanostructures comprise nanorods. In certainembodiments, the nanostructures comprise nanorings. In certainembodiments, the nanostructures comprise nanoplugs. In certainembodiments, the nanostructures comprise nanogratings. In certainembodiments, the wells further comprise a gel material. In certainembodiments, the gel material comprises a hydrogel. In certainembodiments, the solid support comprises a surface of a flow cell.

Also presented herein is a method of making an array, comprisingobtaining a solid support comprising a planar surface, the surfacecomprising a plurality of wells, the wells being separated from eachother by interstitial regions; coating a metal film on the solidsupport; subjecting the metal film to a thermal annealing process,thereby forming a plurality of nanostructures in each of said pluralityof wells. In certain embodiments, the nanostructures are formed of aplasmon resonant material. In certain embodiments, the method furthercomprises polishing the planar surface to substantially removenanostructures from the interstitial regions and to maintain thenanostructures in the wells. In certain embodiments, the method furthercomprises coating at least a portion of the solid support with a gelmaterial, thereby depositing the gel material in a plurality of thewells. In certain embodiments, nanostructures comprise a materialselected from the group consisting of: Gold (Au), Silver (Ag), Tin (Sn)Rhodium (Rh), Ruthenium (Ru), Palladium (Pd), Osmium (Os), Iridium (Ir),Platinum (Pt), Titanium (Ti) and Aluminum (Al), Chromium (Cr), Copper(Cu), p-type doped silicon, n-type doped silicon, and gallium arsenide.The plasmon resonant material may comprise a metallic alloy. Forexample, the plasmon resonant material may comprise Zinc-Indium-TinOxide (ZITO) or Tantalum Oxide (e.g., TaO₅).

Also presented herein is a method of detecting nucleic acids,comprising: providing a solid support comprising a planar surface, thesurface comprising a plurality of wells, the wells being separated fromeach other by interstitial regions; plurality of nanostructures in eachof said plurality of wells; a gel material forming a layer covering theplurality of nanostructures; and a library of target nucleic acids inthe gel material; contacting the solid support with at least onefluorescently labeled probe that binds to the target nucleic acids; anddetecting fluorescent signal on the solid support to distinguish thetarget nucleic acids that bind to the at least one probe. In certainembodiments, the nanostructures are formed of a plasmon resonantmaterial. In certain embodiments, nanostructures comprise a materialselected from the group consisting of: Gold (Au), Silver (Ag), Tin (Sn)Rhodium (Rh), Ruthenium (Ru), Palladium (Pd), Osmium (Os), Iridium (Ir),Platinum (Pt), Titanium (Ti) and Aluminum (Al), Chromium (Cr), Copper(Cu), p-type doped silicon, n-type doped silicon, and gallium arsenide.In certain embodiments, the nanostructures are situated at the bottom ofthe wells. In certain embodiments, the nanostructures are situated alongthe walls of the wells. In certain embodiments, the interstitial regionsare substantially devoid of nanostructures. In certain embodiments, thewells further comprise a gel material. In certain embodiments, the gelmaterial comprises a hydrogel. In certain embodiments, the solid supportcomprises a surface of a flow cell. In certain embodiments, thefluorescently labeled probe comprises a fluorescently labelednucleotide. In certain embodiments, the fluorescently labeled probecomprises a fluorescently labeled oligonucleotide. In certainembodiments, detecting comprises detection of hybridization of anoligonucleotide probe to target nucleic acids in each feature. Incertain embodiments, detecting comprises detection of incorporation of anucleotide or an oligonucleotide probe to target nucleic acids in eachfeature.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cross-section of a portion of a structuredsubstrate formed in accordance with an embodiment.

FIG. 2 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 3 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment that includesnano-imprint lithography (NIL) material.

FIG. 4 illustrates different steps of the method shown in FIG. 3.

FIG. 5 illustrates different steps of the method shown in FIG. 3.

FIG. 6 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment that includes NILmaterial that forms nanostructures.

FIG. 7 illustrates different steps of the method shown in FIG. 6.

FIG. 8A illustrates a perspective view of a nanostructure that may beused with one or more embodiments.

FIG. 8B illustrates a perspective view of a nanostructure that may beused with one or more embodiments.

FIG. 8C illustrates a perspective view of a nanostructure that may beused with one or more embodiments.

FIG. 8D illustrates a perspective view of a nanostructure that may beused with one or more embodiments.

FIG. 8E illustrates a perspective view of a nanostructure that may beused with one or more embodiments.

FIG. 9A illustrates a cross-section of a nanostructure that may be usedwith one or more embodiments.

FIG. 9B illustrates a cross-section of a nanostructure that may be usedwith one or more embodiments.

FIG. 9C illustrates a cross-section of a nanostructure that may be usedwith one or more embodiments.

FIG. 9D illustrates a cross-section of a nanostructure that may be usedwith one or more embodiments.

FIG. 10A illustrates a plan view of a nanostructure that may be usedwith one or more embodiments.

FIG. 10B illustrates a plan view of a nanostructure that may be usedwith one or more embodiments.

FIG. 10C illustrates a plan view of a nanostructure that may be usedwith one or more embodiments.

FIG. 10D illustrates a plan view of a nanostructure that may be usedwith one or more embodiments.

FIG. 11 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 12 illustrates a side view of a deposition step during themanufacture of the structured substrate of FIG. 11.

FIG. 13 is an enlarged cross-sectional view of a reaction cavity duringthe deposition step of FIG. 12.

FIG. 14 illustrates a side view of another deposition step during themanufacture of the structured substrate of FIG. 11.

FIG. 15 is an enlarged cross-sectional view of the reaction cavityduring the deposition step of FIG. 13.

FIG. 16 is an enlarged view of a reaction cavity formed in accordancewith an embodiment.

FIG. 17 is an enlarged view of a reaction cavity formed in accordancewith an embodiment.

FIG. 18 is an enlarged view of a reaction cavity formed in accordancewith an embodiment.

FIG. 19 is a flow chart illustrating a method of detecting lightemissions is accordance with an embodiment.

FIG. 20 is a plan view of a structured substrate having an array ofreaction sites.

FIG. 21 is the plan view of the structured substrate during a firstdetection step.

FIG. 22 is the plan view of the structured substrate during a seconddetection step.

FIG. 23 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 24 is a side view illustrating different steps of the method shownin FIG. 23.

FIG. 25 is a side view illustrating different steps of the method shownin FIG. 23 in which the structured substrate includes separate reactionsites.

FIG. 26 is a scanning electron microscope (SEM) image of a workingsubstrate that was formed using a method similar to the method of FIG.23.

FIG. 27 is a scanning electron microscope (SEM) image of a workingsubstrate that was formed using a method similar to the method of FIG.23.

FIG. 28 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 29 is a side view illustrating different steps of the method shownin FIG. 28.

FIG. 30 is an SEM image of a working substrate that was formed using amethod similar to the method of FIG. 28.

FIG. 31 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 32 is a side view illustrating different steps of the method shownin FIG. 31.

FIG. 33 is an SEM image of a working substrate that was formed using amethod similar to the method of FIG. 31.

FIG. 34 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 35 is a side view illustrating different steps of the method shownin FIG. 34.

FIG. 36 is a flow chart illustrating a method of manufacturing astructured substrate in accordance with an embodiment.

FIG. 37 is a side view illustrating different steps of the method shownin FIG. 36.

FIG. 38 is an SEM image of a working substrate that was formed using amethod similar to the method of FIG. 36.

FIG. 39 is an enlarged SEM image of a working substrate that was formedusing a method similar to the method of FIG. 36.

FIG. 40 is an SEM image of a working substrate that was formed using amethod similar to the method of FIG. 36 after a plasmon resonantmaterial has been provided.

FIG. 41 is an enlarged SEM image of a working substrate that was formedusing a method similar to the method of FIG. 36 after a plasmon resonantmaterial has been provided.

FIG. 42 is a schematic diagram of an imaging system formed in accordancewith an embodiment.

FIG. 43 is a perspective view of a read head including a plurality ofmicrofluorometers formed in accordance with an embodiment.

FIG. 44 is a side view of a structured substrate having a uniformembedded array of nanoparticles in accordance with an embodiment.

DETAILED DESCRIPTION

The present application includes subject matter similar to subjectmatter described in U.S. Provisional Application No. 61/920,244, filedon Dec. 23, 2013, and entitled ENHANCING DNA CLUSTER FLUORSCENCE USINGLOCALIZED SURFACE PLASMON RESONANCE, and in International ApplicationNo. PCT/US2014/072256, filed on Dec. 23, 2014 and entitled STRUCTUREDSUBSTRATES FOR IMPROVING DETECTION OF LIGHT EMISSIONS AND METHODSRELATING TO THE SAME, each of which is incorporated herein by referencein its entirety.

The subject matter of the present application may also be applicablewith or include similar subject matter that is described in U.S. PatentAppl. Publ. Nos. 2014/0242334; 2014/0079923; and 2011/0059865 and U.S.Pat. No. 8,895,249. Each of these publications and the patent isincorporated herein by reference in its entirety.

One or more embodiments set forth herein are configured to directly orindirectly enhance light emissions from an array of reaction sites sothat the light emissions may be detected by, for example, an imagingsystem or device. To this end, embodiments may at least one of increasean intensity of an excitation light experienced by a biologicalsubstance, increase an intensity of the light emissions generated by thebiological substance, and/or control a directionality of the lightemissions so that the light emissions may be detected. The increase inintensity and/or control of the directionality of the light emissionsmay be caused, in part, by one or more nanostructures located at thecorresponding reaction site. The amount of increase may be measuredrelative to an amount of electromagnetic energy that exists at thereaction site without the nanostructure(s).

The array of reaction sites may be disposed along a structuredsubstrate. The structured substrate may be, for example, a flow cellhaving a channel for directing reagents alongside the reaction sites.The light emissions may be detected by an imaging system that mayinclude, for example, an objective lens that scans or sweeps alongsidethe structured substrate to detect the light emissions from the reactionsites. Exemplary systems capable of detecting light emissions from thestructured substrates set forth herein are described in U.S. Appl. Publ.Nos. 2012/0270305 A1 and 2013/0261028 A1, each of which is incorporatedherein by reference in its entirety. Alternatively, the structuredsubstrate may be integrated with an imaging device, such as asolid-state imaging device (e.g., CMOS). In such embodiments, theimaging device may have one or more light sensors that are aligned withreaction sites to capture light emissions from the reaction sites. Suchembodiments are described in U.S. Provisional Application No. 61/914,275and International Application No. PCT/US14/69373, each of which isincorporated herein by reference in its entirety.

A technical effect provided by at least one of the embodiments mayinclude an increased signal intensity from the emitters of thebiological substance. The increase in signal intensity may reduce anerror rate by increasing the likelihood that the signals will bedetected. Another technical effect may include a decrease in signal tonoise ratio that enables faster scan speeds and reduces overall time forconducting a protocol. For instance, with respect tosequencing-by-synthesis technology, faster scan speeds on sequencinginstruments are desired, but faster scan speeds result in fewer photonsbeing collected per cluster on the imaging camera. With fewer photonscaptured, the signal to noise ratio typically decreases and it becomesmore difficult to confidently assign a base. Furthermore on somesequencing instruments, low NA optics result in signals that areinherently larger and dimmer, potentially yielding higher error rates.Embodiments set forth herein may increase the number of photons that arecaptured. Another technical effect for at least some embodimentsincludes a method of manufacturing a structured substrate that is morereliable than at least some known methods and more cost-effective thanat least some known methods.

As used herein, a “biological substance” or “chemical substance”includes biomolecules, samples-of-interest, analytes-of-interest, andother chemical compound(s). A biological substance or chemical substancemay be used to detect, identify, or analyze other chemical compound(s),or function as intermediaries to study or analyze other chemicalcompound(s). In particular embodiments, the biological substance is anucleic acid or, more specifically, a colony of nucleic acids having acommon sequence. In particular embodiments, the biological or chemicalsubstances include a biomolecule. As used herein, a “biomolecule”includes at least one of a biopolymer, nucleoside, nucleic acid,polynucleotide, oligonucleotide, protein, enzyme, polypeptide, antibody,antigen, ligand, receptor, polysaccharide, carbohydrate, polyphosphate,cell, tissue, organism, or fragment thereof or any other biologicallyactive chemical compound(s) such as analogs or mimetics of theaforementioned species.

As another example, a biological or chemical substance may include anenzyme or reagent used in a coupled reaction to detect the product ofanother reaction such as an enzyme or reagent used to detectpyrophosphate in a pyrosequencing reaction. Enzymes and reagents usefulfor pyrophosphate detection are described, for example, in U.S. PatentPublication No. 2005/0244870 A1, which is incorporated herein in itsentirety.

Biological or chemical substances may be naturally occurring orsynthetic and located within a designated area or space. In someembodiments, the biological or chemical substances may be bound to asolid phase or gel material. Biomolecules, samples, and biological orchemical substances may also include a pharmaceutical composition. Insome cases, biomolecules, samples, and biological or chemical substancesof interest may be referred to as targets, probes, or analytes.

Embodiments may be particularly suitable for enhancing emissions fromfluorescently-labeled nucleic acids. By way of example, embodiments mayprovide fluorescent enhancement of DNA clusters in sequencing bysynthesis reactions involving dye-labeled nucleotides. Embodiments mayincrease a signal intensity from flurorescent labels during sequencingby synthesis. The increase in signal intensity may improve overallsequencing performance by reducing sequencing error arising from lowintensity clusters and cluster dropouts during long sequencing runs.

Various embodiments utilize one or more nanostructures to amplifyelectromagnetic energy at a reaction site. For embodiments that utilizea plurality of nanostructures (e.g., two or more nanostructures), theplurality of nanostructures may be referred to as an ensemble amplifier.As used herein, the terms “nanostructure” and “nanoparticle” are usedinterchangeably to refer to a structure having a greatest dimension(e.g, height, width, diameter) in the range of about 1 nm to about 1000nm, including any integer or non-integer value between 1 nm and 1000 nm.In typical embodiments, the nanoparticle is a metallic particle or asilicon particle. In some embodiments, the nanoparticle core is aspherical or nearly spherical particle of 20-200 nm in diameter. In someembodiments the range is about 1 nm to about 50 nm (for example about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nm).

Anisotropic nanostructures (e.g., non-spherical structures) may have alength and a width or, for some embodiments, a diameter. In someembodiments, the length of the anisotropic nanostructure is the greatestdimension of the nanostructure. In some embodiments, the length of ananisotropic nanoparticle is a dimension parallel to the plane of theaperture in which the nanoparticle was produced. In some embodiments,the length of an anisotropic nanoparticle is the dimension perpendicularto the plane of the aperture in which the nanoparticle was produced. Inthe case of anisotropic nanostructures, the nanostructure may have awidth or diameter in the range of about 50 nm to about 750 nm. In otherembodiments, the nanostructure has a width or diameter of about 350 nmor less. In other embodiments, the nanoparticle has a width or diameterof 250 nm or less and in some embodiments, a width or diameter of 100 nmor less. In some embodiments, the width or diameter is between 15 nm to300 nm.

In some embodiments, the nanoparticle has a length of about 10-750 nm.In some embodiments, the nanostructures have a preselected shape and canbe, for example a nanotube, a nanowire, nanosphere, or any shapecomprising the above-described dimensions (e.g., triangular, square,rectangular, or polygonal shape in 2 dimensions, or cuboid, pyramidal,cylindrical, spherical, discoid, or hemispheric shapes in the 3dimensions). Some examples of nanostructures include, for example,bowtie nanoantennae, nanospheres, nanopyramids, nanoshells, nanorods,nanowires, nanorings, nanoplugs, nanogratings and the like. Preformeddimers and trimers of nanostructures can also be loaded into wells andhave the advantage of precisely controlling nanoparticle spacing.

The nanostructures can either be fabricated on a surface or pre-formedand then loaded into the reaction cavities, such as nanowells. Examplesof such structures include plasmonic nanoplugs fabricated at the bottomof nanowells, bowtie and cavity antennas in nanowells, metalnanogratings on which nanowells could be formed, nanostructures reflowedin nanowells or a combination of some or all of the above. One examplewould be a metal nanoplug in a nanowell with nanostructures on the wallsformed through an electron beam evaporation process. Ensemble amplifiersor constructs (dimers, n-mers) may also be positioned within thereaction cavities. Such methods allow for precise subnanometer controlover nanostructure spacings and can be formed on a large scale usingbottom up self-assembly.

The spacing between any two nanostructures on a surface can be anydistance. In some embodiments, the spacing can be a multiple of awavelength of incident light energy, such as a particular emission orexcitation wavelength in fluorescence spectroscopy. The spacing can be,for example, 1 λ, 2 λ, 3 λ, 4 λ, or another multiple of a chosenwavelength (λ) of incident light energy. Thus, using as an example anemission wavelength (λ) of 532 nm, the spacing between nanostructurescan be about 532 nm (1λ), about 1064 nm (2λ), or another multiple of theemission wavelength. In some embodiments, the spacing can be a fractionof a wavelength of incident light energy, such as a particular emissionor excitation wavelength in fluorescence spectroscopy. The spacing canbe, for example, 1λ, ½λ, ⅓λ, ¼λ or another multiple of a chosenwavelength of incident light energy. Thus, using as an example anemission wavelength of (λ) 532 nm, the spacing between nanostructurescan be about 532 nm (1λ), 266 nm (½λ), 133 nm (⅓λ) or another fractionof the emission wavelength.

In some embodiments, the nanostructures may be referred to as “plasmonicnanostructure” or “nanoplasmonic structure.” These terms may be usedinterchangeably and refer to any independent structure exhibitingplasmon resonance characteristic of the structure, including (but notlimited to) both nanostructures, nanostructures and combinations orassociations of nanostructures.

The term “nanoantenna,” as used herein, includes a nanostructure or aplurality of nanostructures (or ensemble amplifier) that acts to amplifyelectromagnetic energy, such as light energy. As used herein, ananoantenna (or ensemble amplifier) does not necessarily exhibit plasmonresonance characteristics. In some embodiments, a nanoantenna does notsubstantially comprise a plasmon resonant material. Thus, in someembodiments, nanoantennas are presented which are made of a non-metalmaterial but which exhibits amplification characteristics ofelectromagnetic energy. Nanostructures presented herein can be of anysuitable shape and size so as to produce the desired energyamplification. Some exemplary shapes of nanoantennas include, forexample, bowtie nanoantennae, nanospheres, nanopyramids, nanoshells,nanorods, nanowires, nanorings, nanoplugs, nanogratings and the like. Itwill be appreciated that any of a number of known methods can besuitable for fabrication and/or deposition of nanoantenna on a solidsupport. Methods for fabrication of nanoantenna are known in the art andinclude, for example, the methods described herein for nanoparticlefabrication and deposition.

The nanostructures can comprise any material suitable for use in themethods and compositions described herein, for example, any type ofmaterial exhibiting surface plasmon resonance (SPR). In certainpreferred embodiments, the nanoparticle comprises a plasmon resonantmaterial. Examples include, but are not limited to, metalnanostructures. For example, the nanostructures can comprise a metalsuch as one or more of Gold (Au), Silver (Ag), Tin (Sn) Rhodium (Rh),Ruthenium (Ru), Palladium (Pd), Osmium (Os), Iridium (Ir), Platinum(Pt), Titanium (Ti) and Aluminum (Al), Chromium (Cr), Copper (Cu), orany other suitable metal. The plasmon resonant material may comprise ametallic alloy. For example, the plasmon resonant material may compriseZinc-Indium-Tin Oxide (ZITO) or Tantalum Oxide (e.g., TaO₅). Thenanostructures can be formed from a single material such as, for examplea single metal. Additionally or alternatively, the nanostructures can beformed from a combination of two or more different materials, such as,for example, two or more metals. For example, the nanostructures cancomprise a metal/metal mixture such as Sn/Au or Ag/Au. Alternatively oradditionally, vertical layered nanostructures, such as multilayerstructures of the metal-insulator-metal type may be applied. Examplesinclude p-type doped silicon, n-type doped silicon, and galliumarsenide. In particular embodiments, the nanostructures may be formedfrom a polymer that is coated by a plasmon resonant material and/or ametallic material.

Formation of nanostructures on a solid support can be performed usingany one of a number of methods known in the art. Nanostructures can beformed using bottom-up self-assembly of plasmonic nanostructures andnano-antennae on the sequencing substrate. For example, any one of anumber of methods for deposition of layers of a material can be used,such as those described by Gaspar et al. (Scientific Reports, 2013, 3,1469), which is incorporated herein by reference. Layer-fabricatingprocesses that may be used to form the nanostructures includephotolithography, etching (e.g., reactive-ion etching), sputtering,evaporation, casting (e.g., spin coating), chemical vapor deposition,electrodeposition, epitaxy, thermal oxidation, physical vapordeposition, and the like. In some embodiments, the nanostructures may beformed using a shadow technique. In some embodiments, the nanostructuresmay be formed using nanolithography, such as nanoimprint lithography(NIL).

In exemplary embodiments described herein, the nanostructures can bepre-formed and mixed in a colloid-like composition with a gel material,which is deposited on a surface. Alternatively or additionally,nanostructures can be first deposited on a surface followed bydeposition of a gel material over the nanostructures. In otherembodiments, a gel material can be deposited on a surface andnanostructures are deposited over the gel material.

In some embodiments, the nanostructures are formed in a well (or concavefeature) of a solid surface. A film of starting material such as Sn/Aucan be deposited on a solid surface containing nanowells, followed bythermal annealing. In some embodiments, thermal annealing can beutilized to promote formation of nanostructures as the film coalescesinto discrete particles. Nanoparticle size can be a function of thestarting film thickness. A further polishing step following the thermalanneal can result in nanostructures only in the wells while leaving theinterstitial regions substantially void of nanostructures.Nanostructures in interstitial regions can be removed through, forexample, chemical and/or mechanical polishing. A distribution ofnanoparticle sizes is observed in each nanowell enabling broad spectrumfluorescence enhancement.

In some embodiments, a nanostructure such as a nanoring can be formedalong the wall of wells (or concave features) on a surface. Thenanostructures can be fabricated using any one of a number ofmethodologies known in the art. For example, Au can be deposited usingsputtered deposition. In an embodiment, conformal deposition of a ˜65 nmAu layer may be followed by a reactive ion etch (ME) process. Theremaining Au layer was located along the walls of the nanowells, formingnanorings in each of the nanowells.

The terms “excitation light” and “light emissions” mean electromagneticenergy and are used to differentiate the source of the electromagneticenergy. Excitation light is generally provided from a light source(e.g., laser) that is positioned a distance away from the reaction site.For example, for embodiments that include reaction cavities, the lightsource may be positioned outside of the reaction cavities. Lightemissions, however, are typically generated by an emitter within or atthe reaction sites. The emitter may be, for example, a fluorophore.Particular embodiments may be configured to amplify electromagneticenergy at any wavelength between 300 nm to 750 nm (e.g., 300 nm, 301 nm,302 nm, 303 nm, 304 nm, 305 nm, 306 nm, 307 nm, 308 nm . . . 745 nm, 746nm, 747 nm, 748 nm, 749 nm, and 750 nm). As used herein, the term“wavelength” shall not be limited to a single wavelength unlessexpressly stated to constitute “a single wavelength” or “only onewavelength”. Instead, the term “wavelength” shall encompass a narrowrange of wavelengths located about a desired or target wavelength (e.g.,532 nm±10 nm, 532 nm±5 nm, 660 nm±10 nm, 660 nm±5 nm), unless explicitlyrecited otherwise.

The nanostructures of each ensemble amplifier may be configured relativeto one another to amplify the electromagnetic energy in a designatedmanner. For example, a distance that separates the nanostructures of acorresponding ensemble amplifier may be based on the electromagneticenergy that is desired to be amplified. The nanostructures of theensemble amplifier may be configured for a particular wavelength (e.g.,narrow band of wavelengths). For example, one or more embodiments may beconfigured to amplify electromagnetic energy having a wavelength of 532nm. One or more embodiments may be configured to amplify electromagneticenergy having a wavelength of 660 nm. In some embodiments, the ensembleamplifiers may be capable of amplifying multiple wavelengths or broaderranges of wavelengths.

One or more embodiments may include ensemble amplifiers thatpreferentially respond to certain polarizations of light. For example, afirst ensemble amplifier may be configured to respond to a firstpolarization light, and a second ensemble amplifier may be configured torespond to a second polarization light. The preferential response may bebased on, for example, a dipole moment of the corresponding ensembleamplifier.

By way of example, when a first ensemble amplifier is illuminated by afirst polarization light, the light emissions provided by the firstensemble amplifier may provide a maximum signal intensity for the firstensemble amplifier. However, when a second ensemble amplifier (which hasa different configuration than the first ensemble amplifier) isilluminated by the first polarization light, the light emissionsprovided by the second ensemble amplifier may be, for example, about 40%or less the maximum signal intensity for the second ensemble amplifier.Likewise, when the second ensemble amplifier is illuminated by thesecond polarization light, the light emissions provided by the secondensemble amplifier may provide a maximum signal intensity for the secondensemble amplifier. However, when the first ensemble amplifier isilluminated by the second polarization light, the light emissionsprovided by the first ensemble amplifier may be, for example, about 40%or less the maximum signal intensity for the first ensemble amplifier.

In many cases, the first and second ensemble amplifiers may beilluminated simultaneously, concurrently, or during the same imagingsequence such that a single image detects light emissions from the firstensemble amplifiers and the second ensemble amplifiers. In suchembodiments, the ensemble amplifiers that preferentially respond to theexcitation light may provide a greater signal intensity than theensemble amplifiers that do not preferentially respond to the excitationlight. A subsequent may then be captured that uses a differentexcitation light.

FIG. 1 is a cross-section of a portion of a structured substrate 100formed in accordance with an embodiment. The structured substrate 100includes a substrate body 102 having an active side 104. The active side104 includes a plurality of reaction sites 106 and a side surface 105that extends between the reaction sites 106. The reaction sites 106 arespaced apart from each other by interstitial regions 118 of thesubstrate body 102. The interstitial regions 118 are areas along theactive side 104 or portions of the substrate body 102 that separate thereaction sites 106 from one another. The side surface 105 extends alongthe interstitial regions 118. In some embodiments, the plurality ofreaction sites 106 form a dense array of reaction sites 106 such thatthe interstitial regions 118 are separated, for example, by less than1000 nm. In particular embodiments, a center-to-center spacing 119between adjacent reaction sites 106 may be less than 1000 nm. Inparticular embodiments, the center-to-center spacing 119 may be lessthan 800 nm, less than 700 nm or, more particularly, less than 500 nm.

In the illustrated embodiment, the interstitial regions 118 include acontinuous, planar side surface 105, but the interstitial regions 118may include non-planar surfaces in other embodiments. The interstitialregions 118 may include a surface material that differs from thematerial of the reaction sites 106 and may functionally isolate thereaction sites 106 from one another. In the illustrated embodiment, onlytwo reaction sites 106 are shown along the active side 104. It should beunderstood, however, that the reaction sites 106 may be part of an arrayof reaction sites that may include hundreds, thousands, or millions ofreaction sites.

In the illustrated embodiment, the reaction sites 106 are cavities and,as such, will hereinafter be referred to as reaction cavities 106. Thereaction cavities 106 are typically concave features that form adepression or indentation along the active side 104. The reactioncavities 106 may be, for example, wells, pits, channels, recesses, andthe like. However, it should be understood that other embodiments mayinclude reaction sites that are not located within cavities. Forexample, the reaction sites may be distributed along a planar surface.Such embodiments are described in U.S. Provisional Application No.61/920,244, which is incorporated herein by reference in its entirety.For example, embodiments that are configured to have ensemble amplifiersthat respond differently to different polarized lights may have reactionareas along a planar surface.

As shown in FIG. 1, a reaction cavity has a cross section that is takenperpendicular to the active side 104. The cross-section may includecurved sections, linear sections, angles, corners. Generally, a reactioncavity need not pass completely through one or more layers. For example,each of the reaction cavities 106 has at least one sidewall 124 thatextends between the active side 104 and a bottom surface 126 of thereaction cavity 106. Both the sidewall 124 and the bottom surface 126are defined by the cavity layer 114. In alternative embodiments, thebase layer 112 (or other layer) may define the bottom 126 of thereaction cavity 106.

The reaction cavities 106 open to the active side 104 such that thereaction cavities 106 are accessible along the active side 104. Forexample, the reaction cavities 106 may be capable of receiving gelmaterial and/or fluid along the active side 104 during manufacture ofthe structured substrate 100 or when the structured substrate 100 isused during analysis. The active side 104 may also receive an excitationlight 108 from a light source (not shown) and/or face an opticalcomponent (not shown), such as an objective lens, that detects lightemissions 110 from the reaction cavities.

The substrate body 102 may be formed from one or more stacked layers. Inthe illustrated embodiment, the substrate body 102 includes a base layer112 and a cavity layer 114. The base layer 112 may be, for example, aglass (SiO₂) wafer. The cavity layer 114 may be a polymer. The substratebody 102, however, may include other layers in alternative embodiments.

As used herein, the term “layer” is not limited to a single continuousbody of material unless otherwise noted. For example, each layer may beformed form multiple sub-layers of the same or different materials.Moreover, each layer may include one or more features of differentmaterials located therein or extending therethrough. The differentlayers may be formed using known layer-fabricating processes, such asphotolithography, etching, sputtering, evaporation, casting (e.g., spincoating), chemical vapor deposition, electrodeposition, epitaxy, thermaloxidation, physical vapor deposition, and the like. One or more layersmay also be formed using nanolithography, such as nanoimprintlithography (NIL). As used herein, the term “working substrate” includesone or more stacked layers in which at least one of the layers is beingprocessed to form a structured substrate from the working substrate.

Each of the reaction cavities 106 may include at least one nanostructure116. The interstitial regions 118 may be substantially devoid ofnanostructures. In other embodiments, however, the nanostructures 116are distributed such that one or more of the nanostructures 116 arelocated within the interstitial regions 118 (as indicated by phantomlines). For example, the nanostructures 116 may be distributed evenly oruniformly along the base layer 112 such that, after the cavity layer 114is formed, the nanostructures 116 are also located or embedded withinthe interstitial regions 118. In some embodiments, the embeddednanostructures 116 within the interstitial regions 118 do not have asubstantial effect on the electromagnetic energy that propagates intothe reaction cavities or the electromagnetic energy that is generatedwithin the reaction cavities. In other embodiments, the embeddednanostructures 116 may have an effect on the electromagnetic energy thatpropagates into the reaction cavities or the electromagnetic energy thatis generated within the reaction cavities.

FIG. 44 illustrates such an example in which a structured substrate 1150includes an array of nanostructures 1154 that are evenly distributedalong a base layer 1152. A cavity layer forms a plurality of cavities1156 in which the nanostructures 1154 form an ensemble amplifier in eachcavity 1156. Such embodiments may reduce the complexity in manufacturingthe structured substrates by not requiring precise alignment of thereaction cavities with the nanostructures.

In the illustrated embodiment of FIG. 1, each of the reaction cavities106 includes a plurality of nanostructures 116. However, it should beunderstood that alternative embodiments may include only a singlenanostructure. The plurality of nanostructures may form an ensembleamplifier, which is hereinafter referred to as an ensemble amplifier120. The ensemble amplifier 120 is positioned within each of thereaction cavities 106 and is configured to at least one of amplifyelectromagnetic energy that propagates into the corresponding reactioncavity or amplify electromagnetic energy that is generated within thecorresponding reaction cavity.

As used herein, an “ensemble of nanostructures” or “ensemble amplifier”includes a plurality of nanostructures that are configured to at leastone of amplify electromagnetic energy that is incident on the discretesite (e.g., reaction cavity) or amplify electromagnetic energy that isgenerated at the discrete site. For instance, the electromagnetic energymay be the excitation light 108 that propagates from an exteriorenvironment and into a reaction cavity 106, wherein the excitation lightis absorbed by an emitter (e.g., fluorophore) that is associated with abiological substance. As another example, the electromagnetic energy maybe light emissions 110 that are emitted from the biological substances.More specifically, after being excited, the fluorophores may emit theelectromagnetic energy (e.g., light emissions 110) that is thenamplified by the ensemble 120 of nanostructures. In some embodiments,the ensemble amplifier 120 may also be referred to as a nanoantenna,because the nanostructures collectively operate to amplify and transmitthe light emissions 110 away from the reaction sites.

An ensemble amplifier may include two or more nanostructures thatoperate in concert to amplify the electromagnetic energy. As describedherein, in some embodiments, an ensemble amplifier may be configured topreferentially amplify a type of electromagnetic energy or, morespecifically, electromagnetic energy having a predetermined wavelength.For example, an ensemble amplifier may have a greater amplificationeffect on light emissions than on excitation light or vice versa.Nonetheless, in some embodiments, an ensemble amplifier may amplify boththe light emissions and the excitation light.

As used herein, when an ensemble amplifier is “configured to amplifyelectromagnetic energy,” each of the nanostructures may have one or morequalities such that the ensemble amplifier collectively operate toamplify the electromagnetic energy. The qualities may include, forexample, a material composition of the nanostructure, a shape of thenanostructure, a size of the nanostructure, and a position of thenanostructure relative to other nanostructures in the ensemble. Forexample, adjacent nanostructures 116 may have a distance 128therebetween that is configured amplify electromagnetic energy that isconfined therebetween. In some embodiments, the resulting amplificationin light emissions may be due to a combination of localized surfaceplasmon resonance and resonant energy transfer processes.

Also shown in FIG. 1, the reaction cavities 106 may include an organicmaterial 122 disposed within the reaction cavities 106. The organicmaterial 122 may cover the nanostructures 116. In some embodiments, theorganic material 122 is configured to immobilize a biomolecule withinthe corresponding reaction cavity. For example, the biomolecule may be anucleic acid. Although not shown in FIG. 1, a passivation layer may beapplied between the organic material 122 and the cavity layer 114 and/orthe nanostructures 116.

In particular embodiments, the organic material 122 includes a gelmaterial, such as a hydrogel. As used herein, the term “gel material” isintended to mean a semi-rigid material that is permeable to liquids andgases. Typically, gel material can swell when liquid is absorbed orreceived by the gel material and can contract when liquid is removedfrom the gel material (e.g., through drying). Exemplary gel materialsinclude, but are not limited to those having a colloidal structure, suchas agarose; polymer mesh structure, such as gelatin; or cross-linkedpolymer structure, such as polyacrylamide, SFA (see, for example, USPat. App. Pub. No. 2011/0059865 A1, which is incorporated herein byreference) or PAZAM (see, for example, U.S. Prov. Pat. App. Ser. No.61/753,833, which is incorporated herein by reference). Particularlyuseful gel material will conform to the shape of a reaction cavity whereit resides. Some useful gel materials can both (a) conform to the shapeof the reaction cavity where it resides and (b) have a volume that doesnot substantially exceed the volume of the reaction cavity where itresides.

In particular embodiments, the organic material 122 has a volume that isconfigured to accommodate only a single analyte such that stericexclusion prevents more than one analyte from being captured or seedingthe reaction cavity. Steric exclusion can be particularly useful forlarge analytes, such as nucleic acids. More specifically, reactioncavities can expose a surface of the organic material (e.g., gelmaterial) having an area that is equivalent to or smaller than adiameter of the excluded volume of target nucleic acids that are to beseeded on the substrate. The excluded volume for a target nucleic acidand its diameter can be determined, for example, from the length of thetarget nucleic acid. Methods for determining the excluded volume ofnucleic acids and the diameter of the excluded volume are described, forexample, in U.S. Pat. No. 7,785,790; Rybenkov et al., Proc. Natl. Acad.Sci. U.S.A. 90: 5307-5311 (1993); Zimmerman et al., J. Mol. Biol.222:599-620 (1991); or Sobel et al., Biopolymers 31:1559-1564 (1991),each of which is incorporated herein by reference. Conditions for stericexclusion are set forth in U.S. Ser. No. 13/661,524 and U.S. Pat. No.7,785,790, each of which is incorporated herein by reference, and can bereadily used for structured substrates of the present disclosure.

In some embodiments, such as embodiments that utilize steric exclusion,a library of target nucleic acids can be delivered to reaction cavitiesthat contain the gel material prior to initiation of an amplificationprocess. For example, target nucleic acids can be delivered to astructured substrate under conditions to seed the gel material in thesubstrate with the target nucleic acids. The structured substrate canoptionally be washed to remove target nucleic acids that do not seed thegel material as well as any other materials that are unwanted forsubsequent processing or use of the structured substrate.

Nonetheless, it will be understood that in other embodiments, the areaof the exposed gel material may be substantially greater than thediameter of the excluded volume of the target nucleic acids that aretransported to the amplification sites. Thus, the area for the featurescan be sufficiently large that steric exclusion does not occur.

Returning to FIG. 1, in some embodiments, the nanostructures 116 areformed along the base layer 112 such that the nanostructures 116 projectfrom the base layer 112 and into the reaction cavities 106. In someembodiments, the nanostructures 116 extend through a portion of thecavity layer 114. In other embodiments, the bottom surface 126 may bedefined by a portion of the base layer 122 such that the nanostructures116 do not extend through the cavity layer 114.

During a protocol in which light emissions are detected by a detector,the light emissions may be generated in response to the excitation light108. In alternative embodiments, the excitation light 108 is notprovided and, instead, the excitation light 108 is generated by emitterscoupled to the biomolecule 129. In some embodiments, a gain field 130exists along one of the nanostructures 116 or between two or morenanostructures 116. The gain field 130 may represent a space where ahigh intensity electric field is created by the nanostructures 116 inresponse to excitation light and/or light emissions. For someapplications, the nanostructures 116 amplify the excitation light 108such that the emitters are more energized by the excitation light andprovide a greater signal intensity for detection. In other applications,the nanostructures 116 do not amplify the excitation light 108, butamplify the light emissions 110 such that the light emissions 110provide a greater signal intensity for detection. In some applications,however, the nanostructures 116 may be capable of amplifying both theexcitation light 108 and the light emissions 110 such that a greaterintensity of the excitation light 108 is experienced by the emitters anda greater intensity of the light emissions 110 is provided by theemitters. Accordingly, embodiments set forth herein may provide agreater signal intensity that is easier to detect by imaging systems ordevices. For example, embodiments set forth herein may provide a greatersignal intensity compared to sites that do not include suchnanostructures.

The present application describes various methods for manufacturing orfabricating structured substrates that may be used to detect or analyzedesignated reactions. At least some of the methods are illustrated inthe figures as a plurality of steps. However, it should be understoodthat embodiments are not limited to the steps illustrated in thefigures. Steps may be omitted, steps may be modified, and/or other stepsmay be added. By way of example, although some embodiments describedherein may include only two layers, other embodiments may include three,four, or more layers. Moreover, steps described herein may be combined,steps may be performed simultaneously, steps may be performedconcurrently, steps may be split into multiple sub-steps, steps may beperformed in a different order, or steps (or a series of steps) may bere-performed in an iterative fashion. In addition, although differentmethods are set forth herein, it should be understood that the differentmethods (or steps of the different methods) may be combined in otherembodiments.

The structured substrates may be formed using one or more processes thatmay, for example, be used to manufacture integrated circuits, duringmicrofabrication, and/or to manufacture nanotechnology. Lithography(e.g., photolithography) is one category of techniques or processes thatmay be used to fabricate the structured substrates described herein. Inparticular embodiments, one or more layers are formed using nanoimprintlithography (NIL). Exemplary lithographic techniques or processes aredescribed in greater detail in Marc J. Madou, Fundamentals ofMicrofabrication and Nanotechnology: Manufacturing Techniques forMicrofabrication and Nanotechnology, Vol. II, 3^(rd) Edition, Part I(pp. 2-145), which is incorporated herein by reference in its entirety.

One or more processes for fabricating structured substrates may alsoinclude subtractive techniques in which material is removed from aworking substrate. Such processes include chemical techniques, such asdry chemical etching, physical/chemical etching, vapor phase etching,chemical machining (CM), anisotropic wet chemical etching, wetphotoetching; electrochemical techniques, such as electrochemicaletching (ECM), electrochemical grinding (ECG), reactive-ion etching(RIE), photoelectrochemical etching; thermal techniques, such as lasermachining, electron beam machining, electrical discharge machining(EDM); and mechanical techniques, such as physical dry etching, sputteretching, ion milling, water-jet machining (WJM), abrasive water-jetmachining (AWJM), abrasive jet machining (AJM), abrasive grinding,electrolytic in-process dressing (ELID) grinding, ultrasonic drilling,focused ion beam (FIB) milling, and the like. The above list is notintended to be limiting and other subtractive techniques or processesmay be used. Exemplary subtractive techniques or processes are describedin greater detail in Marc J. Madou, Fundamentals of Microfabrication andNanotechnology: Manufacturing Techniques for Microfabrication andNanotechnology, Vol. II, 3^(rd) Edition, Part II (pp. 148-384), which isincorporated herein by reference in its entirety.

One or more processes for fabricating structured substrates may alsoinclude additive techniques in which material is added to a workingsubstrate. Such processes include physical vapor deposition (PVD),evaporation (e.g., thermal evaporation), sputtering, ion plating, ioncluster beam deposition, pulsed laser deposition, laser ablationdeposition, molecular beam epitaxy, chemical vapor deposition (CVD)(e.g., atmospheric pressure CVD (APCVD), low pressure CVD (LPCVD), verylow pressure CVD (VLPCVD), ultrahigh vacuum CVD (UHVCVD), metalorganicCVD (MOCVD), laser-assisted chemical vapor deposition (LCVD),plasma-enhanced CVD (PECVD), atomic layer deposition (ALD)), epitaxy(e.g., liquid-phase epitaxy, solid-phase epitaxy), anodization, thermalspray deposition, electroplating, implantation, diffusion, incorporationin the melt, thermal oxidation, laser sputter deposition, reactioninjection molding (RIM), self-assembled monolayers (SAMs), sol-geladdition, spin coating, polymer spraying, polymer dry film lamination,casting, plasma polymerization, silk screening, ink jet printing,mechanical microspotting, microcontact printing, stereolithography ormicrophotoforming, electrochemical forming processes, electrodeposition,spray pyrolysis, laser beam deposition, electron beam deposition, plasmaspray deposition, micromolding, LIGA (which is a German acronym forx-ray lithography, electrodeposition, and molding), compression molding,and the like. The above list is not intended to be limiting and otheradditive techniques or processes may be used. Exemplary additivetechniques or processes are described in greater detail in Marc J.Madou, Fundamentals of Microfabrication and Nanotechnology:Manufacturing Techniques for Microfabrication and Nanotechnology, Vol.II, 3^(rd) Edition, Part III (pp. 384-642), which is incorporated hereinby reference in its entirety. As used herein, the term “exemplary,” whenused as an adjective, means serving as an example. The term does notindicate that the object to which it modifies is preferred.

FIG. 2 is a flowchart illustrating a method 200 of manufacturing astructured substrate. The method 200 includes providing, at 202, a baselayer (or working substrate) having a base side. The base layer may beonly a single layer of material or include one or more sub-layers. Thebase side may have a planar surface that is configured to have anotherlayer deposited directly thereon. However, it is contemplated that thebase side may include non-planar features prior to being combined withother layers. In particular embodiments, the base layer includes a glass(SiO₂) wafer, but other materials may be used.

The method 200 may also include forming, at 204, an array ofnanostructures along the base side of the base layer. The forming, at204, may include multiple processing steps. For example, the forming, at204, may include providing (e.g., through deposition, growing, oranother additive technique) a feature layer along the base side of thebase layer. The forming, at 204, may include shaping (e.g., throughetching or another subtractive technique) a sub-layer of the base layerto form the nanostructures. The sub-layer may also be referred to as afeature layer as the nanostructures may be formed from the sub-layer.The feature layer may include a material that is capable of being shapedinto individual features that may at least partially form a basis of thenanostructures. The material may include a pure material (e.g., gold) oran alloy of material. The feature layer may also include multiplesub-layers of material (e.g., gold and chrome) that are stackedalongside each other. Optionally, one or more of the materials is aplasmon resonant material.

In particular embodiments, the forming, at 204, includes etching thefeature layer to form nanobodies. The nanobodies may be arranged insub-arrays or sets in which each sub-array (or set) may become anensemble amplifier. In other embodiments, the nanobodies are distributedevenly or uniformly throughout the base layer, such as shown in FIG. 44.In such embodiments, some of the nanobodies will be embedded whileothers will be disposed within the reaction cavities.

In some embodiments, the nanobodies formed from the etching process mayconstitute, without further modification, nanostructures that arecapable amplifying electromagnetic energy. In other embodiments,however, further processing steps may be necessary to form thenanostructures. For example, the feature layer may comprise a polymer(or other material that is not a plasmon resonant material) that may beshaped to form nanobodies for constructing the nanostructures. A thinlayer or film may be subsequently added to exterior surfaces of thenanobodies to form the nanostructures. Yet still in other embodiments,the nanostructures may be locally deposited at select locations. Themethod 500 (FIG. 11) describes such a process.

The method 200 also includes forming, at 206, a cavity layer along thebase side of the base layer. The cavity layer is configured to includethe reaction cavities. For embodiments that do not include reactioncavities, the cavity layer may be referred to as a site layer. As usedherein, the phrase “along the base side” or “along the base layer”includes the cavity layer being in direct contact with the base layer orincludes the cavity layer being separated from the base layer by one ormore intervening layers. As used herein, spatially relative terms, suchas “top,” “above,” “below,” and the like, are used herein for ease ofdescription to distinguish one element or feature from another. Thespatially relative terms do not require that the structured substratehave a particular orientation with respect to gravity during use oroperation. For example, the active side of the structured substrate mayface in a direction that is opposite the gravitational force directionin some embodiments. Alternatively, the active side of the structuredsubstrate may face in the same direction as the gravitational forcedirection in other embodiments. The uppermost surface, such as the sidesurface that liquid flows along during operation, may be referred to asthe top surface regardless of the orientation of the structuredsubstrate with respect to gravity.

The forming, at 206, may include providing a cavity layer that isconfigured to have an array of reaction cavities. The forming, at 206,may include multiple steps. In some embodiments, the cavity layerincludes pre-formed reaction cavities. Each of the reaction cavities maybe aligned with a corresponding sub-array or set of nanostructures(e.g., two or more nanostructures). Optionally, the cavity layer may beetched to remove portions of the cavity layer and expose thenanostructures within the corresponding reaction cavities.

In other embodiments, the reaction cavities may be shaped while thecavity layer is positioned above and coupled to the base layer. Forexample, NIL material may be deposited along the base side of the baselayer after the nanostructures are formed and cover the nanostructures.The NIL material may be deposited using, for example, a spin coatingtechnique or by depositing droplets along the base side. The NILmaterial may comprise a material that is capable of being imprintedusing the NIL technique. For example, the NIL material may comprise apolymer. The NIL material may then be imprinted or stamped with a mold(also called template) having a pattern of features that form thereaction cavities in the NIL layer. In some embodiments, the mold istransparent to allow ultraviolet (UV) or visible light to propagatetherethrough. In such embodiments, the NIL material may comprise aphotocurable polymer that is cured by the UV or visible light while themold is pressed into the NIL material. Accordingly, the NIL material maycure (e.g., harden) to form the reaction cavities. This process may beidentical or similar to step-and-flash imprint lithography (SFIL). Inother embodiments, the NIL material may be cured by application ofthermal energy and/or pressure. The NIL techniques and like processesare described in Marc J. Madou, Fundamentals of Microfabrication andNanotechnology: Manufacturing Techniques for Microfabrication andNanotechnology, Vol. II, 3^(rd) Edition, Part I (pp. 113-116) and Lucaset al., “Nanoimprint Lithography Based Approach for the Fabrication ofLarge-Area, Uniformly Oriented Plasmonic Arrays” Adv. Mater. 2008, 20,1129-1134, each of which is incorporated herein by reference in itsentirety.

Each of the reaction cavities may be aligned with a correspondingsub-array of nanostructures. The NIL material may be preferentiallyetched to expose the plurality of nanostructures within thecorresponding reaction cavity. Regardless of the method ofmanufacturing, the sub-array of nanostructures may form an ensembleamplifier of the corresponding reaction cavity. The ensemble amplifieris configured to at least one of amplify electromagnetic energypropagating into the corresponding reaction cavity or amplifyelectromagnetic energy generated within the corresponding reactioncavity.

Optionally, the method 200 may also include providing, at 208, anorganic material within the reaction cavities. The organic material maycover the nanostructures. In some embodiments, the organic material isprovided across the active side, including the interstitial regions. Theorganic material may then be removed by polishing the active side. Afterthe active side is polished, each of the reaction cavities may includecorresponding organic material that is separated from other organicmaterial of other reaction cavities. In particular embodiments, theorganic material is a gel material, such as those described herein(e.g., PAZAM, SFA or chemically modified variants thereof, such as theazidolyzed version of SFA (azido-SFA).

The method 200 may also include additional steps, such as preparingsurfaces of the structured substrate to interact with the fluids andsamples of a designated protocol. As another example, the method 200 mayinclude mounting, at 210, a flow cover to the active side of the cavitylayer. The flow cover may define a flow channel between the flow coverand the active side. Embodiments that include flow covers are describedin U.S. Provisional Application No. 61/914,275 and InternationalApplication No. PCT/US14/69373, each of which is incorporated herein byreference in its entirety.

FIG. 3 illustrates a flowchart of a method 220 of manufacturing astructured substrate 280 (shown in FIG. 5). The method 220 is describedwith reference to FIGS. 4 and 5. The method 220 may include one or moresteps that are similar or identical to the steps of method 200 (FIG. 2).The method 220 includes providing, at 222, a base layer (or workingsubstrate) 240 having a base side 242. The method 220 also includesforming, at 224, an array 244 of nanostructures 246 along the base side242. For example, a feature layer 245 may be provided to the base side242 (e.g., through a deposition process) of the base layer 240. Thefeature layer 245 may be etched to form the array 244 of nanostructures246. The array 244 may include sub-arrays 248 of the nanostructures 246.Also shown in FIG. 4, adjacent sub-arrays 248 are separated by a spacing250 along the base side 242. In other embodiments, however, the featurelayer 245 is etched such that the array of nanostructures 246 extendsuniformly across the base layer 240 (see, e.g., FIG. 44). In suchembodiments, some of the nanostructures 246 may be covered or embeddedwhen the structured substrate is complete while other nanostructures 246may be disposed within corresponding reaction cavities. By using auniform array of nanostructures 246, it may not be necessary (or may beless difficult) to align the reaction cavities during manufacturing. Yetin other embodiments, the nanostructures 246 are formed across the baselayer 240 in a generally random manner.

Each sub-array 248 may include a plurality of the nanostructures 246that collectively form an ensemble amplifier when the structuredsubstrate 280 (FIG. 5) is fully formed. For example, the nanostructures246 of each sub-array 248 may be sized, shaped, and positioned relativeto each other such that the nanostructures 246 amplify electromagneticenergy. In the illustrated embodiment, the nanostructures 246 areillustrated as upright posts that have a common shape and size. However,it should be understood that the nanostructures 246 may have differentshapes in other embodiments. Furthermore, the nanostructures 246 of asingle sub-array 248 are not required to have an identical shape and/oran identical size.

At 226, a NIL material 252 may be provided along the base side 242 ofthe base layer 240. The NIL material 252 may cover the array 244 of thenanostructures 246. The NIL material 252 may be a viscous material suchthat the NIL material 252 surrounds and fills empty spaces between thenanostructures 246. The NIL material 252 may comprise, for example, apolymer. In the illustrated embodiment, the NIL material 252 is providedas a NIL layer along the base side 242. In other embodiments, the NILmaterial may be provided as an array of droplets that, when compressedduring an imprinting operation, effectively cover at least portions ofthe base side 242.

At 228, an array 254 of reaction cavities 256 may be imprinted into theNIL material 252. The imprinting, at 228, may include applying a mold258 to the NIL material 252. The mold 258 may have a non-planar side 260that includes a pattern of features. The features are sized, shaped, andpositioned relative to each to shape the NIL material 252 in apredetermined manner such that the reaction cavities 256 are formed.When the mold 258 is applied to the NIL material 252, a stacked assembly262 is formed that includes the mold 258, the NIL material 252, thenanostructures 246, and the base layer 240.

The imprinting, at 228, may also include curing the NIL material 252 tosolidify the shape of the NIL material 252. For example, the curingprocess may include applying a UV light or visible light 264 to thestacked assembly 262. The NIL material 252 may comprise a photopolymerthat is capable of solidifying after being exposed to the UV or visiblelight 264. However, alternative methods of solidifying or curing the NILlayer 252 may be used. For example, thermal energy (e.g., heat) orpressure may be applied to the NIL material 252 to solidify the NILmaterial 252 and form the reaction cavities 256.

With respect to FIG. 5, after the curing process, the NIL materialbecomes a solidified NIL layer 253 having the array 254 of reactioncavities 256. The solidified NIL layer 253 may constitute a cavitylayer, such as the cavity layer 114 (FIG. 1), that includes the reactioncavities 256. Each reaction cavity 256 may be aligned with acorresponding sub-array 248 of the nanostructures 246 such that thereaction cavity 256 is positioned above the corresponding sub-array 248.As shown in FIG. 4, the nanostructures 246 may be positioned within afill region 266 of the solidified NIL layer 253. The fill region 266includes the nanostructures 246 surrounded by the solidified material ofthe NIL layer 253. At this stage, the fill region 266 may define abottom surface 268 of the reaction cavity 256. Also shown, at thisstage, the reaction cavities 256 may be separated by interstitialregions 270, which separate the reaction cavities 256. For embodimentsin which the nanostructures 246 are uniformly spaced along the baselayer 240, one or more nanostructures 246 may be located within theinterstitial regions 270.

The method 220 may also include removing, at 230, the fill regions 266to expose at least portions of the nanostructures 246 within thecorresponding reaction cavities 256. For example, a preferential etchingprocess may be applied to remove the material of the NIL layer 253 thatsurrounds the nanostructures 246 without substantially damaging orremoving the nanostructures 246. During the removing, at 230, the bottomsurface 268 of each reaction cavity 256 is lowered such that the bottomsurface 268 approaches the base layer 240. In some embodiments, the NILlayer 253 within the fill regions 266 may be etched entirely such thatthe base layer 240 forms at least a portion of the bottom surface 268.In other embodiments, similar to the structured substrate 100 of FIG. 1,a portion of the NIL layer 253 may remain after the etching process. Insuch embodiments, the nanostructures 246 may extend through the NILlayer 253 (or cavity layer). During the removing, at 230, theinterstitial regions 270 may also be etched, as indicated, such that aheight of the interstitial regions 270 relative to the base layer 240 isreduced. The height is reduced from 271A to 271B.

As described above, the nanostructures 246 within each reaction cavity256 may form an ensemble amplifier 272 of the corresponding reactioncavity 256. The ensemble amplifier 272 is configured to at least one ofamplify electromagnetic energy propagating into the correspondingreaction cavity or amplify electromagnetic energy generated within thecorresponding reaction cavity.

The structured substrate 280 is shown at the bottom of FIG. 5. Thestructure substrate 280 includes an active side 282 and has the reactioncavities 256 and the interstitial regions 270 that separate the reactioncavities 256. Optionally, the method 200 may include providing, at 232,an organic material 274 within the reaction cavities 256. Prior toproviding the organic material 274, the working substrate may beprocessed for receiving the organic material 274. For example, apassivation layer (e.g., tantalum oxide or the like) and a layer ofsilane may be provided onto the passivation layer. Both the passivationlayer and the silane layer may cover the nanostructures 246. Theproviding, at 232, may include spin coating the organic material ontothe working substrate. However, other additive techniques may also beused. Optionally, the working substrate having the passivation layer,the silane layer, and the organic material may be incubated.

As shown in FIG. 5, the organic material 274 may cover thenanostructures 246 in the reaction cavities 256. In some embodiments,the organic material 274 is provided across the entire active side 282such that the organic material 274 covers surfaces of the interstitialregions 270. The organic material 274 may then be removed by polishingthe active side 282. After the active side 282 is polished, each of thereaction cavities 256 may include organic material 274 therein that isseparated from organic material 274 in adjacent reaction cavities 256.The organic material 274 within each reaction cavity 256 surrounds thenanostructures 246 of the ensemble amplifier 272. The organic material274 may be configured to support and/or hold a biological or chemicalsubstance that is capable of providing light emissions, such asdye-labeled nucleic acids.

FIG. 6 is a flowchart illustrating a method 300 of manufacturing orfabricating a structured substrate. In some embodiments, the method 300includes steps that are similar or identical to the steps of the methods200 (FIGS. 2) and 220 (FIG. 3). Different stages of the method 300 areillustrated in FIG. 7. The method 300 may include providing, at 302, abase layer (or working substrate) 320 having a base side 322 andproviding, at 304, NIL material 324 along the base side 322 of the baselayer 320. In some embodiments, the NIL material 324 may be provided asa NIL layer. In other embodiments, the NIL material 324 may be providedas separate droplets along the base side 322.

The method 300 may also include imprinting, at 306, the NIL material324. After imprinting, the NIL material 324 may be a solidified NILlayer 324 having a base portion 326 (indicated by the dashed line) andan array 328 of nanobodies 330 that project from the base portion 326.In some embodiments, the nanobodies 330 are arranged to form sub-arrays,but in other embodiments the nanobodies 330 are uniformly distributedalong the base layer 320, such as the nanostructures 1154 shown in FIG.44. The base portion 326 extends between adjacent nanobodies 330. Thenanobodies 330 may have a variety of shapes. In the illustratedembodiment, the nanobodies 330 are elongated posts that project awayfrom the base portion 326 of the NIL layer 324. In alternativeembodiments, the base portion 326 is not formed after imprinting.Instead, only the nanobodies 330 may be formed after imprinting.

The method 300 may also include providing, at 308, a plasmon resonantlayer 334 along the NIL material 324 and, in particular, the nanobodies330. The providing, at 308, may also be referred to as depositing orgrowing. In some embodiments, the plasmon resonant layer 334 may be athin film or coating. The providing, at 308, may be executed using oneor more additive techniques. For example, the providing, at 308, mayinclude at least one of PECVD, ALD, evaporation, sputtering, spincoating, or the like. The plasmon resonant layer 334 includes a plasmonresonant material (e.g., gold, silver, silicon, and the like) thatcovers the nanobodies 330. Accordingly, nanostructures 332 may be formedin which each nanostructure 332 includes a respective nanobody 330 and aportion of the plasmon resonant layer 334 that covers or surrounds therespective nanobody 330.

Optionally, the method 300 may include providing, at 310, a passivationlayer 336. The passivation layer 336 is configured to protect theunderlying layers, such as the plasmon resonant layer 334, from damageduring use of the structured substrate.

At 312, the method 300 may include forming a cavity layer 338 along theoperative side 341 that includes a plurality of reaction cavities 340.In some embodiments, the cavity layer 338 may be formed using, forexample, a NIL technique in which a NIL material is imprinted and curedto form the reaction cavities 340. In FIG. 7, only a single reactioncavity 340 is shown, but it should be understood that an array ofreaction cavities 340 may be formed.

Also optionally, the method 300 may include providing, at 314, a silanelayer (not shown). The silane layer may be configured to facilitatecoupling between an organic material and/or biological or chemicalsubstances. By way of example, the providing, at 314, may beaccomplished by vapor deposition. In some embodiments, the silane layermay be provided after or before other processing steps. At this stage,the base layer 320, the NIL material 324, the plasmon resonant layer334, the passivation layer 336, and the optional silane layer may form aworking substrate 339 having an operative side 341.

If the cavity layer 338 is formed using a NIL process, empty spacebetween the nanostructures 332 may be filled with the NIL material 324.As described above with respect to the method 220, the NIL material 324may be removed through preferential etching. After the NIL material isremoved, an ensemble amplifier 342 of the nanostructures 332 may beformed within the corresponding reaction cavity 340. At 316, an organicmaterial may be provided to the reaction cavities 340.

In the illustrated embodiment, the cavity layer 338 is formed using aNIL process. However, it should be understood that the cavity layer 338may be formed using other additive and/or subtractive processes, such asthose described above.

FIGS. 8-10 illustrate different nanostructures that may be implementedwith one or more embodiments. However, the nanostructures shown in FIGS.8-10 are exemplary only and are not intended to be limiting. Othernanostructures may be used in alternative embodiments. In FIGS. 8A-8D,the nanostructures are located within corresponding cylindrically-shapedreaction cavities. In other embodiments, the reaction cavities may havea different shape. For example, a cross-section of the reaction cavitymay be oval-shaped, square-shaped, rectangular, other polygonal shape,or the like. Yet in other embodiments, the nanostructures may be locatedalong a planar surface.

FIG. 8A is a perspective view of a nanoplug 402 in a reaction cavity404, which may also be referred to as a nanowell. The nanoplug 402 maycomprise gold (Au). In the illustrated embodiment, the nanoplug 402 iscentrally located within the reaction cavity 404, but it may have otherpositions in other embodiments. FIG. 8B is a perspective view of abowtie antenna 406 that may be use within one or more embodiments. Thebowtie antenna 406 includes two separate nanostructures 408 that aretriangular in shape and point to each other with a small gaptherebetween. The bowtie antenna 406 may form an ensemble amplifier.FIG. 8C illustrates a nanograting 410 in a reaction cavity 412 thatincludes a series of spaced-apart beams 411. The nanograting 410 may beformed in a lower layer and subsequently exposed when the reactioncavity 412 is formed above the nanograting 410. As shown, thenanograting 410 is not confined within the reaction cavity 412 andextends beyond the wall of the reaction cavity 412. FIG. 8D illustratesa plurality of nanoparticles 414 disposed within a reaction cavity 416.The nanoparticles 414 may be distributed in random locations within thereaction cavity 416. The nanoparticles 414 may be formed, for example,through a reflow or deposition process. FIG. 8E illustrates a dimer 420and a trimer 422. The dimer 420 and the trimer 422 may be disposedwithin alone in a single reaction cavity (not shown) without othernanostructures disposed therein. Alternatively, the dimer 420 and trimer422 may share a common reaction cavity. Optionally, the dimer 420 andtrimer 422 are not disposed within reaction cavities and, instead, aredistributed along a planar surface (not shown).

FIGS. 9A-9D illustrate side cross-sections of reaction cavities havingnanostructures disposed therein. The reaction cavities may be, forexample, cylindrical or rectangular-shaped. In FIG. 9A, a reactioncavity 430 is shown that includes a plurality of nanostructures 432. Thenanostructures 432 are posts that may be cylindrical or square-shaped.In FIG. 9B, a reaction cavity 434 is shown that includes a plurality ofnanostructures 436. The nanostructures 436 may be conical or pyramidal.In FIG. 9C, a reaction cavity 438 is shown that includes a plurality ofnanostructures 440. Each of the nanostructures 440 may be conical orpyramidal and have a particle portion 442 disposed at a top of thenanostructure 440. In FIG. 9D, a reaction cavity 444 is shown thatincludes a plurality of nanostructures 446. The nanostructures 446constitute sidewalls that face each other.

FIGS. 10A-10D illustrate plan views of reaction cavities having one ormore nanostructures disposed therein. More specifically, FIG. 10Aillustrates a nanoring 450 that surrounds a central axis 452. Thenanoring 450 is circular in FIG. 10A, but may have other shapes (e.g.,polygonal) in other embodiments. FIG. 10B illustrates five posts 454that are positioned relative to one another. FIGS. 10C and 10D showbowtie antennas 456, 458, respectively. The bowtie antennas 456, 458 areconfigured to preferentially respond to different polarizations oflight.

In each of FIGS. 8A-8C, 9A-9D, and 10B-10D, the nanostructures may beconfigured to form a corresponding ensemble amplifier that isorientation dependent such that the ensemble amplifier preferentiallyresponds to a polarized light of a designated orientation. Such ensembleamplifiers may be referred to as polarized amplifiers. For example, theensemble amplifiers may be configured to have a dipole moment that isessentially parallel to an excitation light of a designatedpolarization. The amount of light emissions provided by reactioncavities having such polarized amplifiers is dependent upon thepolarization of the excitation light.

In other embodiments, the ensemble amplifiers may be configured topreferentially respond to light emissions of a predetermined wavelength.For example, if the emitters provide light emissions that are equal toor near the predetermined wavelength, the ensemble amplifiers mayamplify the light emissions. However, if the emitters provide lightemissions that are not equal to or near the predetermined wavelength,the ensemble amplifiers may only partially amplify the light emissionsor amplify the light emissions by a negligible amount.

FIG. 11 is a flowchart illustrating a method 500 of manufacturing orfabricating a structured substrate. The method may include performingone or more additive or subtractive techniques, such as those describedabove. In some embodiments, the method 500 includes steps that aresimilar or identical to the steps of the methods 200 (FIG. 2), 220 (FIG.3), and 300 (FIG. 6). Different stages of the method 500 are illustratedin FIGS. 12-15. The method 500 includes providing, at 502, a workingsubstrate 522 having an operative side 523. The working substrate 522may represent an unfinished or incomplete structured substrate. Theworking substrate 522 may be similar to one or more of the base layersand/or other working substrates described herein. For example, theworking substrate 522 may include one or more structures (e.g., layers,features, and the like) that have been provided using the additive andsubtractive techniques described above.

The operative side 523 has a non-planar contour that includes a sidesurface 524 and an array of receiving cavities 526 that open to the sidesurface 524. In the illustrated embodiment, the side surface 524 isplanar between the receiving cavities 526. The side surface 524 is notrequired to be planar, however, and may include projections or otherfeatures. As described herein, embodiments may utilize the non-planarcontour of the operative side 523 to form nanostructures at desiredlocations along the operative side 523, such as within the receivingcavities 526.

Each of the receiving cavities 526 has an opening 528 along the sidesurface 524. The side surface 524 includes interstitial regions thatextend between and separate adjacent openings 528. Each of the receivingcavities 526 extends a depth 530 from the corresponding opening 528 intothe working substrate 522 to a bottom surface 532. As shown in FIG. 12,the receiving cavities 526 coincide with an array plane 525. Morespecifically, the array plane 525 may intersect each of the receivingcavities 526. In some embodiments, the array plane 525 extends parallelto the side surface 524 and/or one or more of the layers that form theworking substrate 522. For example, a glass wafer 527 may form a bottomlayer of the working substrate 522. The array plane 525 may extendparallel to the glass wafer 527.

The method 500 may also include positioning, at 504, the workingsubstrate 522 in a receiving orientation 529, which may also be referredto as the first receiving orientation in some embodiments. The method500 may also include directing, at 506, a deposition stream 536 onto theoperative side 523 of the working substrate 522. The deposition stream536 may be provided by a deposition source 540. The deposition stream536 includes a feature material 542 (shown in FIG. 13). In particularembodiments, the deposition stream 536 is provided in a substantiallylinear manner (e.g., in one direction along an axis). As such, thedirecting operation at 506 may be characterized as line-of-sightdeposition. For example, the deposition source 540 is an electron beamevaporation system. However, it is contemplated that other line-of-sightdeposition sources may be used.

In particular embodiments, the feature material 542 is a plasmonresonant material that accumulates along the operative side 523 todirectly form nanostructures that are configured to amplifyelectromagnetic energy as set forth herein. However, in otherembodiments, the feature material 524 may not be a plasmon resonantmaterial. In such embodiments, the feature material 524 may be used toindirectly form nanostructures. For example, the feature material 542may form nanobodies and a plasmon resonant material may be subsequentlydeposited over the nanobodies to form nanostructures capable ofamplifying electromagnetic energy.

In FIG. 12, the deposition stream 536 appears as a plurality of separatestreams. In some embodiments, the deposition stream 536 may be a singlestream that is scanned along the operative side 523. For example, thedeposition source and/or the working substrate 522 may be moved relativeto one another so that the deposition stream 536 moves along theoperative side 523. In other embodiments, multiple deposition streams536 may be applied concurrently. Optionally, a mask having apertures maybe positioned between the deposition source 540 and the workingsubstrate 522 to block the deposition stream 536 during portions of thedeposition operation.

The directing, at 506, may include directing the deposition stream 536at a non-orthogonal angle 544 with respect to the working substrate 522when the working substrate 522 is in the receiving orientation 529. Forexample, the directing, at 506, may include directing the depositionstream 536 at the non-orthogonal angle 544 with respect to the arrayplane 525. Additionally or alternatively, the non-orthogonal angle 544may be with respect to the side surface 524. The non-orthogonal angle544 may be, for example, between 5° and 85°. In some embodiments, thenon-orthogonal angle 544 is between 10° and 75°. In particularembodiments, the non-orthogonal angle 544 is between 15° and 60°.

FIG. 13 is an enlarged side view of an exemplary receiving cavity 526during the directing, at 506 (i.e., during a deposition process), whenthe working substrate 522 is in the receiving orientation 529 (FIG. 12).As shown, the receiving cavity 526 is defined by a cavity surface 548.The cavity surface 548 may be a single surface having a curved contouror separate surfaces that are joined at, for example, corners. Forinstance, the cavity surface 548 includes a wall surface 550 and thebottom surface 532 that includes a maximum depth of the receiving cavity526. The wall surface 550 may be a single circular or curved surface.Alternatively, the wall surface 550 may include multiple surfaces thatare joined at, for example, corners of the receiving cavity 526. Thewall surface 550 extends from an opening edge 554 that intersects theside surface 524 to a corner 556 formed with the bottom surface 532.

The directing, at 506, is configured to utilize the non-planar contourof the operative side 523 of the working substrate 522 to block portionsof the deposition stream 536 from entering the receiving cavities 526and to allow other portions of the deposition stream 536 to enter thereceiving cavities 526. For example, if multiple deposition streams areconcurrently incident on the operative side 523, then the non-planarcontour would block one or more of the deposition streams from enteringthe receiving cavities 526. If a single deposition stream is scanned(e.g., moved) along the operative side 523, then the non-planar contourmay block the deposition stream for a portion of the scan time. In thismanner, the feature material 542 may accumulate in selected areas alongthe cavity surface 548.

For example, in FIG. 13, the working substrate 522 is positioned in thereceiving orientation 529 relative to a linear path of the depositionstream 536. In the receiving orientation 529, a shadow area 558 isformed along the cavity surface 548. The shadow area 558 is indicated inFIG. 13 as a solid line that extends along the cavity surface 548. In anexemplary embodiment, the shadow area 558 includes at least a portion ofthe wall surface 550 and at least a portion of the bottom surface 532.

In the receiving orientation 529, an incident area 560 is also formedalong the cavity surface 548. In the illustrated embodiment, theincident area 560 includes at least a portion of the wall surface 550and at least a portion of the bottom surface 532. The incident area 560is indicated in FIG. 13 as a dashed line that extends along the cavitysurface 548 and also along the side surface 524.

During the deposition process, the feature material 542 of thedeposition stream 536 is permitted to pass through the opening 528 andaccumulate along the incident area 560 within the receiving cavity 526.The feature material 542, however, does not accumulate along the shadowarea 558. Instead, the side surface 524 blocks or obstructs thedeposition stream 536 from entering the receiving cavity 526 and beingincident on the shadow area 558. Accordingly, after the depositionprocess, at 506, one portion of the cavity surface 548 (e.g., theincident area 560) includes feature material 542 thereon, but anotherportion (e.g., the shadow area 558) is devoid of the feature material542.

In some embodiments, the method 500 includes repeating the positioning,at 504, and the directing, at 506. For example, the working substrate522 may be re-positioned in a different second receiving orientation andanother deposition stream may be provided onto the working substrate522. In alternative embodiments, the rotation may occur while thedeposition stream 562 is provided to the working substrate 522.

FIGS. 14 and 15 illustrate the second deposition process. In FIGS. 14and 15, the deposition stream is referenced as a deposition stream 562.The deposition stream 562 may include a feature material 564 that isidentical to or different from the feature material 542 (FIG. 13). InFIGS. 14 and 15, the working substrate 522 is in a second receivingorientation 572 that is different from the first receiving orientation529. The working substrate 522 may be moved in any amount or directionto the second receiving orientation 572. For example, relative to theworking substrate 522 in FIG. 12, the working substrate 522 may berotated about a central axis 570 that extends substantially parallel tothe path of the deposition stream 562. The working substrate 522 may berotated, for example, +/- 45, 90, 135, 180 relative to the firstreceiving orientation 520 to be positioned in a second receivingorientation 572. The working substrate 522 may also be rotated aboutother axes that are perpendicular to the central vertical axis 570. Forexample, the working substrate 522 may be rotated about an axis that isperpendicular to the central axis 570 to increase or decrease thenon-orthogonal angle 544.

As shown in FIG. 15, in the second receiving orientation 572, a secondshadow area 566 (indicated by solid line) and a second incident area 568(indicated by dashed line) are formed in each receiving cavity 526. Insome embodiments, the second shadow area 566 may at least partiallyoverlap with the first incident area 560 (FIG. 13), and the secondincident area 568 may at least partially overlap the first shadow area558 (FIG. 13). In the illustrated embodiment, the second shadow area 566includes at least a portion of the wall surface 550 and at least aportion of the bottom surface 532. In the illustrated embodiment, thesecond incident area 568 includes at least a portion of the wall surface550 and at least a portion of the bottom surface 532.

During the second deposition process, the feature material 564 of thedeposition stream 562 is permitted to pass through the opening 528 andaccumulate along the incident area 568 within the receiving cavity 526.In some embodiments, if the feature material 542 is located along theincident area 568, then the feature material 564 may accumulate over thefeature material 542. In some embodiments, if the feature material 542is not located along the incident area 568, then the feature material564 may accumulate directly over the incident area 568 of the cavitysurface 548.

The feature material 564, however, does not accumulate along the secondshadow area 566. Instead, the side surface 524 blocks or obstructs thedeposition stream 562 from entering the receiving cavity 526.Accordingly, after the second deposition process a portion of the cavitysurface 548 includes the feature material 564 therealong, but the otherportion is devoid of the feature material 564. The portion devoid of thefeature material 564, however, may already include the feature material542.

In some embodiments, the feature material 542 may form onenanostructure, and the feature material 564 may form anothernanostructure. Optionally, the positioning, at 504, and the directing,at 506, may be repeated one or more times to build nanobodies and/ornanostructures within the receiving cavities 526. Collectively, thenanostructures within each receiving cavity 526 may form an ensembleamplifier as described herein.

After depositing the feature material(s) along the operative side 523,extraneous or unwanted feature material(s) along the side surface 524may be removed, at 508. For example, the side surface 524 may bepolished to remove the feature material(s) and/or another subtractivetechnique may be applied to remove the feature material(s). Optionally,at 510, an organic material (not shown), such as the gel materialdescribed herein, may be provided. The organic material may cover thenanostructures in the receiving cavities 526. Optionally, prior toadding the organic material, the nanostructures may be coated with aplasmon resonant material and/or passivation layer as described herein.At 512, a flow cell may be mounted to the working substrate.

FIG. 16 is a plan view of a reaction cavity 600 having an ensembleamplifier 602 that includes nanostructures 604, 606. In someembodiments, the reaction cavity 600 and the ensemble amplifier 602 maybe manufactured, for example, using the method 500 (FIG. 11). Forexample, the nanostructure 604 may be formed during a first depositionprocess, and the nanostructure 606 may be formed during a seconddeposition process after re-positioning the working substrate. Thenanostructures 604, 606 are located on opposite sides of the reactioncavity 600 and oppose each other with a gap 608 therebetween.

In some embodiments, the ensemble amplifier 602 is a polarized amplifierthat is configured to preferentially respond to electromagnetic energyof a predetermined polarization. For example, the ensemble amplifiers602 may be configured to have a dipole moment μ that may be essentiallyparallel to an excitation light of a predetermined polarization. Whenthe electromagnetic energy of the predetermined polarization is incidenton the ensemble amplifier 602, the reaction cavity 600 and/or theensemble amplifier 602 may preferentially respond to the excitationlight. More specifically, the signal intensity of the light emissionsprovided by the reaction cavity 600 is greater when the dipole momentμof the ensemble amplifier 602 is parallel to the polarization of theexcitation light compared to when the dipole moment μ of the ensembleamplifier 602 is not parallel to the polarization of the excitationlight. In other words, the signal intensity of the light emissionsprovided by reaction cavity 600 in response to the excitation light isdependent upon the polarization of the excitation light.

FIG. 17 is an enlarged view of a reaction cavity 610 having an ensembleamplifier 612 that includes nanostructures 604-607. In some embodiments,the reaction cavity 610 and the ensemble amplifier 612 may bemanufactured, for example, using the method 500 (FIG. 11) and multipledifferent receiving orientations. For example, the nanostructure 614 maybe formed during a first deposition process, the nanostructure 615 maybe formed during a second deposition process, the nanostructure 616 maybe formed during a third deposition process, the nanostructure 617 maybe formed during a second deposition process. In some embodiments, thematerial that forms the nanostructures 614-617 is the same material. Inother embodiments, however, one or more of the nanostructures 614-617may include a different material.

The ensemble amplifier 612 may have two dipole moments μ₁ nd μ₂. Thenanostructures 614, 616 are located on opposite sides of the reactioncavity 610, and the nanostructures 615, 617 are located on oppositesides of the reaction cavity 610. In such embodiments, the ensembleamplifier 612 may preferentially respond to two different polarizationsof excitation light.

FIG. 18 is an enlarged view of a reaction cavity 620 having an ensembleamplifier 622 that includes nanostructures 624-626. In some embodiments,the reaction cavity 620 and the ensemble amplifier 622 may bemanufactured, for example, using the method 500 (FIG. 11) and multipledifferent receiving orientations. For example, the nanostructure 624 maybe formed during a first deposition process, the nanostructure 625 maybe formed during a second deposition process, the nanostructure 626 maybe formed during a third deposition process. In an exemplary embodiment,the material that forms the nanostructures 624-626 are differentmaterials. In other embodiments, however, the material may be the same.

The ensemble amplifier 622 may have two dipole moments μ₃ and μ₄. Forexample, a portion of the nanostructures 624 is located opposite thenanostructure 625, and another portion of the nanostructures 624 islocated opposite the nanostructure 626. In such embodiments, theensemble amplifier 622 may preferentially respond to two differentpolarizations of excitation light. It should be noted, however, that thepreferential responses may not be equal. For example, the signalintensity provided when the dipole moment μ₃ is parallel to thepolarization of the excitation light may be different from the signalintensity provided when the dipole moment μ₄ is parallel to thepolarization of the excitation light. The difference in signal intensitymay be caused by the different materials used to form the nanostructures625 and 626.

Although not shown in FIGS. 16-18, one or more embodiments may includeindividual nanostructures that are formed from two or more plasmonresonant materials. Moreover, one or more of the individualnanostructures may be formed during multiple depositions processes. Forexample, a portion of the nanostructure 624 may comprise gold (Au) andanother portion of the nanostructure 624 may comprise silver (Ag).

FIG. 19 is a flowchart illustrating a method 640. The method 640 may be,for example, a method of conducting an assay protocol in which asequence of fluidic and imaging steps occur. In some embodiments, themethod 640 is a method of detecting light emissions. The method 640 isdescribed in reference to FIGS. 20 and 21, which illustrate an array 662of reaction sites 664. The method 640 includes, at 642, providing astructured substrate having an array 662 of reaction sites 664. Thestructured substrate may be, for example, similar or identical to thestructured substrates described herein. In the illustrated embodiment,the reaction sites 664 are reaction cavities, but it should beunderstood that other embodiments may include reaction areas distributedalong, for example, a common planar surface. The reaction sites 664 maybe similar to, for example, the reaction cavity 600 (FIG. 16). Each ofthe reaction sites 664 includes an ensemble amplifier 668 that is apolarized amplifier. The ensemble amplifiers 668 are configured topreferentially respond to electromagnetic energy having a predeterminedpolarization. The ensemble amplifiers 668 preferentially respond byamplifying the electromagnetic energy.

The array 662 of reaction sites 664 include first and second sub-arrays670, 672, which are shown in FIGS. 21 and 22, respectively. The firstsub-array 670 includes reaction sites 664A having ensemble amplifiers668A, and the second sub-array 672 includes reaction sites 664B havingensemble amplifiers 668B. The ensemble amplifiers 668A of the firstsub-array 670 are configured to preferentially respond to a firstpolarized excitation light. The ensemble amplifiers 668B of the secondsub-array 672 are configured to preferentially respond to a differentsecond polarized excitation light. The first and second polarizedexcitation lights may differ by, for example, about 90°. However, thedifference may be smaller or greater depending upon the application andconfiguration of the ensemble amplifiers.

In the illustrated embodiment, the reaction sites 664A and 664B haveeffectively the same ensemble amplifier 668. More specifically, each ofthe ensemble amplifiers 668 includes a pair of nanostructures that arepositioned relative to one another in the same manner. For example, thenanostructures have the same shape and directly oppose each other.However, the ensemble amplifiers 668A and 668B have different first andsecond orientations such that the ensemble amplifiers 668A have a dipolemoment μ₅ and the ensemble amplifiers 668B have a dipole moment μ₆. Thedipole moments μ₅ and μ₆ differ by about 90°, but may differ by otheramounts in other embodiments.

Turning to FIG. 21, the method 640 includes illuminating, at 644, thearray 662 of reaction sites 664 with a first polarized excitation light(or an excitation light having a first polarization). In someembodiments, the entire array 662 is illuminated when the array 662 isilluminated with the first polarized excitation light. Morespecifically, each of the first and second sub-arrays 670, 672 may beilluminated. In other embodiments, however, only portions of the array662 is illuminated when the array 662 is illuminated with the firstpolarized excitation light. For example, only the first sub-array 670may be illuminated.

At 646, the light emissions from the first sub-array 670 may bedetected. Each of the reaction sites 664A in the first sub-array 670 isconfigured to amplify the excitation light having the firstpolarization. In some embodiments, the amplification may cause a greaterintensity of light emissions from a biomolecule or analyte (e.g.,nucleic acids) located at or within the reaction site 664A. For example,if the biomolecule or analyte includes a plurality of fluorescentlabels, the fluorescent labels may experience a greater intensity ofexcitation light and, consequently, provide a greater response to theexcitation light. It should be noted that, for some embodiments, one ormore of the reaction sites 664A may not include a biomolecule or analytehaving the fluorescent labels. For example, if the desired reaction didnot occur at or within the reaction site 664A, the reaction site 664 maynot have fluorescent labels capable of responding.

For illustrative purposes, FIG. 21 more clearly shows the firstsub-array 670 of reaction sites 664A. In some embodiments, the reactionsites 664B (indicated by circles in FIG. 21) may provide a partialresponse when excited by the excitation light of the first polarization.For example, the reaction sites 664B may emit a signal intensity thatis, 40% or less than the average signal intensity provided by thereaction sites 664A that have the designated emitters (e.g., fluorescentlabels). More specifically, if the average signal intensity from thereaction sites 664B having the designated emitters is Y, then thereaction sites 664B having the designated emitters may provide, at most.0.4Y. In such embodiments, the imaging system may identify thoselocations as providing an insufficient or inadequate response. Inparticular embodiments, the reaction sites 664B having the designatedemitters may emit a signal intensity that is, on average, 30% or less,20% or less, or 10% or less than the average signal intensity providedby the reaction sites 664A.

With respect to FIG. 22, the method 640 also includes illuminating, at648, the array 662 of reaction sites 664 with the second polarizedexcitation light. As described above, in some embodiments, the entirearray 662 is illuminated when the array 662 is illuminated with thesecond polarized excitation light. In other embodiments, however, onlyportions of the array 662 is illuminated when the array 662 isilluminated with the second polarized excitation light. For example,only the second sub-array 672 may be illuminated.

At 650, the light emissions from the second sub-array 672 may bedetected. Each of the reaction sites 664B in the second sub-array 672 isconfigured to amplify the excitation light having the secondpolarization. The amplification of the excitation light may cause agreater intensity of light emissions from a biomolecule or analyte(e.g., nucleic acids) located at the reaction site 664B. For example, ifthe biomolecule or analyte includes a plurality of fluorescent labels,the fluorescent labels may experience a greater intensity of excitationlight and, consequently, provide a greater response to the excitationlight. As described above, it should be noted that one or more of thereaction sites 664B may not include a biomolecule or analyte having thefluorescent labels in some embodiments.

For illustrative purposes, FIG. 22 more clearly shows the secondsub-array 672 of reaction sites 664B. In some embodiments, the reactionsites 664A (indicated by circles in FIG. 22) may provide a partialresponse when excited by the excitation light of the secondpolarization. For example, the reaction sites 664A may emit a signalintensity that is, 40% or less than the average signal intensityprovided by the reaction sites 664B that have the designated emitters(e.g., fluorescent labels). More specifically, if the average signalintensity from the reaction sites 664B having the designated emitters isZ, then the reaction sites 664A having the designated emitters mayprovide, at most. 0.4Z. In such embodiments, the imaging system mayidentify those locations as providing an insufficient or inadequateresponse. In particular embodiments, the reaction sites 664A having thedesignated emitters may emit a signal intensity that is, on average, 30%or less, 20% or less, or 10% or less than the average signal intensityprovided by the reaction sites 664B.

The embodiment described with respect to FIGS. 19-22 may be suitable forhigh density arrays. For example, returning to FIG. 20, the reactionsites 664 form rows 691 and columns 692. The reaction sites 664 within acommon row 691 may have a center-to-centering spacing 684, and thereaction sites 664 within a common column 692 may have acenter-to-center spacing 686. In the illustrated embodiment, theensemble amplifiers 668A, 668B are positioned relative to each otherwithin the array 662 such that each reaction site 664A (or ensembleamplifier 668A) is closer to reaction sites 664B (or ensemble amplifiers668B) than to another reaction site 664A (or ensemble amplifier 668A).For example, the center-to-center spacing 684 between adjacent ensembleamplifiers 668 in a common row may be about × measured in, for example,nanometers (nm), and the center-to-center spacing 686 between adjacentensemble amplifiers 668 in a common column may be about ×. Adjacentreaction sites 664 having the same polarized amplifier may have acenter-to-center spacing 688. As shown, the center-to-center spacing 688is greater than each of the center-to-center spacings 684, 686. Forexample, the center-to-center spacing 688 may be about 1.4×. In otherembodiments, the center-to-center spacing 688 may be at least about1.2×, at least about 1.3×, at least about 1.5×. at least about 1.6×, atleast about 1.7×, at least about 1.8×, at least about 1.9×, or at leastabout 2×. By way of example, the center-to-center spacings 684, 686 maybe about 350 nm, and the center-to-center spacing 688 may be about 500nm. In other embodiments, the center-to-center spacings 684, 686 may beabout 200 nm, 250 nm, 300 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm,800 nm, or more. Although the center-to-center spacings 684, 686 areessentially equal in the illustrated embodiment, the center-to-centerspacings 684, 686 may differ in other embodiments.

Accordingly, in some embodiments, neighboring reaction sites, such asthose that are in the same column or in the same row, may have acenter-to-center spacing that is not optically resolvable by the imagingsystem. If such neighboring reaction sites were imaged simultaneously,each of the reaction sites would emit fluorescence simultaneously. Theimaging system may not be able to differentiate between theseneighboring reaction sites. In contrast, the imaging system may be ableto differentiate between neighboring reaction sites having differentensemble amplifiers. In such an arrangement, reaction sites with thesame ensemble amplifier may have a larger center-to-center spacing. Thislarger center-to-center spacing may be a distance that is within theimaging resolution of the system. Thus, by including ensemble amplifiersthat respond to different polarizations of excitation light, some of thereaction sites (first reaction sites) would be imageable with a firstscan in which the excitation light has a first polarization and otherreaction sites (second reaction sites) would be imageable with a secondscan in which the excitation light has a second polarization. The firstand second reaction sites may be positioned relative to one another sothat the center-to-center spacings between first reaction sites isincreased and the center-to-center spacings between second reactionsites is increased.

For some embodiments, the method 640 may include repeating steps 644,646, 648, and 650 a plurality of times. As an example, the sequence ofsteps 644, 646, 648, 650 may be repeated 20 times, 40 times, 60 times,80 times, 100 times, 120 times, 140 times, 160 times, 180 times, 200times, or more. The method 640 may be part of a sequencing by synthesis(SBS) protocol as described herein in which the sequence of steps 644,646, 648, 650 is performed after the incorporation of labelednucleotides to clusters or colonies of nucleic acids. For example, priorto performing the sequence of steps 644, 646, 648, 650, a liquidcomprising labeled reagents (e.g., nucleotides) may be directed alongthe reaction sites 664 to allow the nucleotides to be added to thenucleic acids. A subsequent wash step may be directed along the reactionsites 664 to remove the unincorporated reagents. After theunincorporated reagents are removed, the sequence of steps 644, 646,648, 650 may be performed to detect the light emissions and determinewhich nucleotide was incorporated by the clusters. After detecting thelight emissions, the labels may be removed and another cycle ofincorporating and detecting the nucleotides may begin.

Although FIGS. 19-22 illustrate an embodiment in which there are onlytwo sub-arrays, it should be understood that other embodiments mayinclude multiple sub-arrays. For example, alternative embodiments mayhave three, four, five, or more different polarized amplifiers.

FIGS. 23-41 illustrate different methods of manufacturing or fabricatingstructured substrates that include nanostructures. In some cases, thenanostructures may form ensemble amplifiers as described above. Thestructured substrates set forth below and elsewhere in the presentapplication may be used to conduct designated chemical reactions foranalyzing biological or chemical substances. In particular embodiments,the structured substrates may be used during an SBS protocol.

For various embodiments, such as those described above and below, it isunderstood that one or more sites (e.g., cavities or localized areas ona surface) of a structured substrate may include nanoparticles that arenot suitably positioned relative to one another such that the lightemissions and/or excitation light can be amplified. Nonetheless, themethods set forth herein may be capable of providing structuredsubstrates in which a significant number of sites may be capable ofamplifying the electromagnetic energy. For example, in some embodiments,more than 50% of the sites may have nanoparticles that are capable ofamplifying the electromagnetic energy. In some embodiments, more than60% or 70% of the sites may have nanoparticles that are capable ofamplifying the electromagnetic energy. In particular embodiments, morethan 80% or 90% of the sites may have nanoparticles that are capable ofamplifying the electromagnetic energy.

FIG. 23 is a flowchart illustrating a method 700 of manufacturing orfabricating a structured substrate. The method 700 may includeperforming one or more additive or subtractive techniques, such as thosedescribed above. The method 700 is illustrated, separately, in FIGS. 24and 25. The method 700 may be similar to the other methods ofmanufacturing described herein and may include one or more steps of theother methods. In an exemplary embodiment, the method 700 includesproviding, at 702, a base layer 712 having a base side 714 andproviding, at 704, a feature layer 716 along the base side 714. In FIG.24, the feature layer 716 is a continuous, planar layer that issubstantially devoid of recess and extends throughout the base side 714.In FIG. 25, however, the feature layer 716 is non-planar and includesdesignated recesses 718. The designated recesses 718 may be formedthrough, for example, NIL as described above. The feature layer 716 mayinclude a resin. For example, the feature layer 716 may comprise EVG orother material that is suitable for NIL.

At 706, the method 700 may include forming nanobodies 720 usingreactive-ion etching (ME). RIE may be used to remove material having adesignated chemistry. For example, the feature layer 716 may comprise acarbon-based material. The RIE may include a chemically-reactive plasmathat is configured to remove the material of the feature layer 716 whenapplied thereto. For instance, the RIE may include using an oxygenplasma to remove portions of the carbon-based material of the featurelayer 716. It should be understood, however, that the above is only oneexample and other materials may be used for the RIE or the feature layer716.

As shown in FIGS. 24 and 25, the ME may provide an irregular surfacealong the feature layer 716 that forms peaks 722 and recesses or troughs724. The peaks 722 and recesses 724 may define the nanobodies 720. Thepeaks 722 may be separated from adjacent peaks by a peak-to-peakdistance 726. The peak-to-peak distances 726 and sizes of the nanobodies726 appear irregular in FIGS. 24 and 25. In some embodiments, the RIEprocess may be configured to achieve an average peak-to-peak distance726. In some embodiments, the RIE process may be configured to provide amajority of the peak-to-peak distances 726 within a designated range.Various parameters may be selected to achieve a desired result. Forexample, the parameters may include the material for the feature layer716, the material or type of ME, the etch time, the thickness of thefeature layer 716, and/or the pitch or distribution of the recesses 718.

As shown in FIG. 25, the nanobodies 720 may form groups or ensembles 730that are separated from other groups 730 by an area 732 of the base side714. Each of the groups 730 includes a plurality of nanobodies 720. Thelocations of the areas 732 may correlate to the locations of therecesses 718 prior to ME. More specifically, the reduced thickness ofthe feature layer 716 at the recesses 718 results in the ME processremoving all of the material of the feature layer 716 such that theareas 732 are exposed. However, as shown in FIG. 24, the feature layer716 may form a distribution of the nanobodies 720 across an entirety ofthe base side 714.

At 708, the etched feature layer 716 may be coated with a plasmonresonant material 734, such as gold (Au). At 710, a passivation layer736 (e.g., Ta₂O₅) may be coated onto the plasmon resonant material 734.As shown, the irregular surface of the feature layer 716 may cause thepassivation layer 736 to form peaks 738 and recesses 740. The peaks 738and recesses 740 may form nanostructures 742 in which adjacent peaks 738may correspond to adjacent nanostructures 742. As described herein,electromagnetic energy may be amplified by the adjacent nanostructures742.

Each of the peaks 738 may be separated by a peak-to-peak distance 744.In the illustrated embodiment, the peak-to-peak distance 744 appearsirregular or non-uniform. However, a majority of the peak-to-peakdistances 744 may be within a designated range. For example, more than75% of the peak-to-peak distances 744 may be between 0.5× and 1.5×,wherein X is greater than or equal to 1 nm and less than or equal to1000 nm. By way of example, × may be less than 900 nm, 800 nm, 700 nm,600 nm, or 500 nm. In particular embodiments, × may be less than 400 nm,350 nm, 300 nm, 350 nm, or 200 nm. In more particular embodiments, × maybe less than 150 nm, 100 nm, 75 nm, 60 nm, or 50 nm. Yet in moreparticular embodiments, × may be less than 40 nm, 30 nm, 20 nm, 15 nm,or 10 nm. As a specific example, more than 75% of the peak-to-peakdistances 726 may be between 1 nm and about 50 nm. More specifically,more than 75% of the peak-to-peak distances 726 may be between 1 nm andabout 25 nm. In certain embodiments, more than 75% of the peak-to-peakdistances 726 may be between 1 nm and about 10 nm.

As shown in FIG. 25, the groups 730 may be used to form reaction sitesor islands 732 in which each reaction site 732 is a localized group ofnanostructures 742 that are formed from an irregular surface of theuppermost layer (e.g., passivation layer 736, plasmon resonant layer734, or feature layer 716). The reaction sites 732 may be separated fromone another by areas 746. Although not shown, the method 900 may alsoinclude providing an organic material (e.g., hydrogel), as describedabove, along the nanostructures 742.

FIGS. 26 and 27 illustrate SEM images of structured substrates 750, 760,respectively, that were formed using a process that is similar to themethod 700. In particular, a feature layer was provided and shaped toinclude recesses, which were similar to the recesses 718 (FIG. 25), thatwere defined by interstitial regions. After an RIE process, the recessesformed the areas 752 and 762. However, the interstitial regions formedthe nanobodies 754, 764. The nanobodies 754 in FIG. 26 are smaller thanthe nanobodies 764 in FIG. 27. The sizes of the nanobodies 754, 764 andspaces 756, 766, respectively, between adjacent nanobodies may be basedon various parameters, such as the material of the feature layer,duration of the RIE process, thickness of the feature layer, and thetype of RIE plasma used. After the nanobodies 754, 765 are coated with aplasmon resonant material, it is contemplated that dye-labeledbiological or chemical substances may be positioned within the spaces756, 766 and the adjacent nanoparticles may form an ensemble amplifier.

FIG. 28 is a flowchart showing a method 800 of manufacturing orfabricating a structured substrate. The method 800 may includeperforming one or more additive or subtractive techniques, such as thosedescribed above. The method 800 is illustrated with respect to FIG. 29.The method 800 include providing, at 802, a working substrate 812 havinga plurality of cavities or recesses 813 separated by interstitialregions 815. The working substrate 812 may be formed using one or moreprocesses described herein. For example, the working substrate 812 mayinclude fused silica, although other materials may be used. As anotherexample, the working substrate 812 may be formed through an NIL process.At 804, a coating material 814 may be provided onto the workingsubstrate 812. The coating material 814 may include a resin or otherviscous material 816 (e.g., high viscosity hydrogel) havingnanoparticles 818 dispersed therein. The nanoparticles 818 may includegold particles or other plasmon resonant material particles. Inparticular embodiments, the coating material 814 may be spin coated ontothe working substrate 812 such that the coating material 814 existswithin the cavities 813 and along the interstitial regions 815. Theproviding, at 804, may also include thermally annealing (e.g., baking)the coating material 814 onto the working substrate 812.

At 806, the coating material 814 may be selectively etched to remove theformerly viscous material 816 and reveal the nanoparticles 818.Optionally, the method may include removing nanoparticles 818 from theinterstitial regions 815. For example, the working substrate 812 may bepolished. At 808, a passivation layer 820 may be applied over thenanoparticles 818 and the interstitial regions 815. For example, Ta2O5may be sputtered onto the nanoparticles 818 and the interstitial regions815. Accordingly, a structured substrate 822 may be provided thatincludes a plurality of cavities 813 that each have a plurality ofnanoparticles 818 therein. The nanoparticles 818 may be relativelydispersed within the cavities 813 such that two or more of the pluralityof nanoparticles 818 are separated by a distance that is suitable foramplifying light emissions and/or excitation light.

FIG. 30 is an SEM image of a structured substrate 824 that was formed inaccordance with the method 800. As shown, the structured substrate 824includes a base layer or working substrate 826 having a plurality ofcavities 828. Each of the cavities 828 includes a plurality ofnanoparticles 830 deposited therein.

FIG. 31 is a flowchart illustrating a method 850 of manufacturing orfabricating a structured substrate. The method 850 is illustrated withrespect to FIG. 32. The method 850 may include performing one or moreadditive or subtractive techniques, such as those described above. Insome embodiments, the method 850 includes steps that are similar oridentical to the steps of the method 500 (FIG. 11) or one of the othermethods described herein. The method 850 includes providing, at 852, aworking substrate 862 having an operative side 864. The workingsubstrate 862 may be similar to one or more of the working substratesand/or other working substrates described herein. For example, theworking substrate 862 may include one or more structures (e.g., layers,features, and the like) that have been provided using the additive andsubtractive techniques described above.

The operative side 864 has a non-planar contour that includes a sidesurface 866 and an array of receiving cavities 868 that open to the sidesurface 866. In the illustrated embodiment, the side surface 866 isplanar between the receiving cavities 868. The method 850 may includedirecting, at 854, a deposition stream 870 onto the operative side 864of the working substrate 862. The deposition stream 870 is directed at anon-orthogonal angle with respect to the operative side 864. Thedeposition stream 870 may be provided by a deposition source (notshown). The deposition stream 870 includes a feature material 872, suchas a plasmon resonant material. In particular embodiments, thedeposition stream 872 is provided in a substantially linear manner(e.g., in one direction along an axis). As such, the directing operationat 854 may be characterized as line-of-sight deposition. For example,the deposition source may be an electron beam evaporation system.However, it is contemplated that other line-of-sight deposition sourcesmay be used.

The receiving cavities 868 include bottom surfaces 874. The featurematerial 872 is deposited along the bottom surfaces 874 and surfaces ofthe interstitial regions 875. However, due to the non-orthogonal angleof the directed deposition and the shadow effect, the feature material872 in each receiving cavity 868 may be localized closer to one end orside of the receiving cavity 868 as shown in FIG. 32.

After directing, at 854, a deposition stream at a non-orthogonal angleonto the working substrate 862, the method 850 may include transforming,at 856, the deposited layer of the feature material 872 intonanoparticles 876. For example, the deposited layer of the featurematerial 872 may be thermally annealed or reflowed to transform thelayer into nanoparticles 876. During thermal annealing, the depositedlayer may be heated (e.g., 400° C.) such that the deposited layercoalesces into discrete nanoparticles. The nanoparticle size can be afunction of the starting thickness of the deposited layer.

Optionally, at 858, the deposited layer may be removed from theinterstitial regions 875 and, at 860, a passivation layer 861 (e.g.,Ta₂O₅) may be sputtered onto the nanoparticles 876 and the workingsubstrate 862. Accordingly, a structured substrate 880 may be providedthat includes a plurality of nanoparticles 876 that are grouped togetherwithin each of the receiving cavities 868.

FIG. 33 is an SEM image of a structured substrate 890 that was formed inaccordance with the method 850 (FIG. 31). As shown, the structuredsubstrate 890 includes a working substrate (or cavity layer) 892 havinga plurality of cavities 894. Each of the cavities 894 includes aplurality of nanoparticles 896 deposited therein. As shown, thenanoparticles 896 are localized or grouped closer to one side of thereceiving cavity 894.

FIG. 34 is a flowchart illustrating a method 900 of manufacturing orfabricating a structured substrate. The method 900 is illustrated withrespect to FIG. 35. The method 900 may include performing one or moreadditive or subtractive techniques, such as those described above. Forexample, the method 900 includes providing, at 902, a working substrate(or base layer) 912 having a substrate side 915. The working substrate912 may be, for example, a glass wafer or a layer of fused silica. At904, a NIL material 914 is provided along the substrate side 915 of theworking substrate 912. For example, the NIL material 914 may bedeposited along the working substrate 912 using a spin coating techniqueor by depositing a designated pattern of droplets along the substrateside 915. The NIL material 914 may comprise a curable material 917 thatis capable of being imprinted using the NIL technique, such as apolymer. The NIL material 914 also includes a plurality of nanoparticles916 that are dispersed within the curable material 917.

At 906, the NIL material 914 may be imprinted to form a non-planarfeature layer 918. For example, a mold (not shown) having a mold side orsurface with a predetermined contour may be pressed into the ML material914 such that the NIL material 914 is sandwiched between the mold andthe substrate side 915. The NIL material 914 may then flow into thevoids of the mold and take a complementary shape of the mold. The NILmaterial 914 may then be cured or activated by light, pressure, and/orheat to form the non-planar feature layer 918.

The non-planar feature layer 918 includes a plurality of recesses 920that are separated by interstitial regions 922. In the illustratedembodiment, a portion of the NIL material 914 remains between a bottomsurface 924 of the recess 920 and the substrate side 915. In otherembodiments, however, the mold may be configured to reduce or minimizethe amount of NIL material 914 that exists between the bottom surface924 and the substrate side 915. As shown in FIG. 35, a greater number ofnanoparticles 916 exist within the interstitial regions 922 than thenumber of nanoparticles 916 that exist within the portion that extendsbetween the bottom surface 924 and the substrate side 915.

At 908, the NIL material 914 may be preferentially or selectively etchedto remove the curable material 917 of the NIL material 914. For example,an RIE process may be applied to the feature layer 918 to remove thecurable material 917 and reveal or expose the nanoparticles 916 alongthe substrate side 915. Optionally, a passivation layer (not shown) maybe applied onto the working substrate 912 to cover the nanoparticles916. Accordingly, a structured substrate 930 may be provided.

As shown in FIG. 35, the structured substrate 930 includes dense regions932 and sparse regions 934. The dense regions 932 include a greaterdensity of the nanoparticles 916 compared to the sparse region 934. Thegreater density of the nanoparticles 916 is caused by the greater numberof nanoparticles 916 that existed within the interstitial regions 922.As such, the locations of the dense regions 932 correlate to thelocations of the interstitial regions 922 of the feature layer 918. Thesparse regions 934 correspond to the location of the recess 920. Thedensity of the dense regions 932 and/or sparse regions 934 may be basedon the contour or shape of the mold (or contour of the feature layer918) and the density of nanoparticles 916 dispersed within the NILmaterial 916. In some embodiments, the dense regions 932 may includenanoparticles 916 that are positioned on top of each other to form athree-dimensional structure. Optionally, the sparse regions 934 may beremoved through a subsequent etching process to form substantially blankareas 936. The blank areas 936 may separate the dense regions 932 ofnanoparticles 916. The dense regions 932 may correspond to reactionsites along the substrate side 915. As described herein, thenanoparticles 916 may form nanostructures and, in some cases, ensembleamplifiers that at least one of amplify electromagnetic energy thatpropagates into the corresponding reaction site or amplifyelectromagnetic energy that is generated within the correspondingreaction site.

FIG. 36 is a flowchart showing a method 950 of manufacturing orfabricating a structured substrate. The method 950 is illustrated withrespect to FIG. 37. The method 950 may include performing one or moreadditive or subtractive techniques, such as those described above. Forexample, the method 950 includes forming, at 952, a plurality ofnanobodies 964 along a base layer 962. In an exemplary embodiment, thebase layer 962 may comprise a glass wafer or fused silica (SiO2). Thebase layer 962 may include other sub-layers, such as tantalum oxide,which may be used to form the nanobodies 964. In some embodiments, thenanobodies 964 are formed using photolithographic processes. However, itis contemplated that the nanobodies 964 may be formed using otherprocesses. For example, the nanobodies 964 may be formed using NILprocesses, such as those described above. The nanobodies 964 may have aheight 966 of, for example, about 100-1000 nm, but other heights may beused. In particular embodiments, the nanobodies 964 are posts that mayhave a greatest cross-sectional dimension of 100-500 nm. Thecross-sections may be, for example, circular or square-shaped.

At 954, a plasmon resonant material 968 (e.g., gold (Au)) may bedeposited along the base layer 962 and nanobodies 964. For instance, theplasmon resonant material may be directionally deposited using electronbeam evaporation. The plasmon resonant material forms a plasmon resonantlayer 968 having a designated thickness. The thickness may be, forexample, between 10 and 200 nm or, more particularly, between about 50and 150 nm. However, other thicknesses may be used. At 956, the workingsubstrate may be subjected to a thermal annealing process to transformthe plasmon resonant layer 968 into nanoparticles 970 along thenanobodies 964. For example, the working substrate may be heated to 500°C. for about 10 minutes. At 958, a passivation layer 972 (e.g., Ta₂O₅)may be applied. The passivation layer 972 may be applied, for example,through a sputter coating process. In some embodiments, the method 950may also include providing another layer over the passivation layer and,optionally, forming recesses or cavity from the added layer.

FIG. 38 illustrates a first SEM image of an array 979 of nanoposts 980that were formed using a method similar to the method 950. FIG. 39 is asecond SEM image of the array 979 of nanoposts 980 at a greatermagnification than the magnification of FIG. 38. The nanoposts 980 areformed from fused silica. The nanoposts 980 are cylindrical and have aheight of about 800 nm and a diameter of about 350 nm. FIG. 40illustrates a first SEM image of the array 979 after 100 nm of gold (Au)was directionally deposited onto the nanoposts 980 and thermallyannealed for about 10 minutes at 500° C. FIG. 41 is a second SEM imageof the array 979 after thermally annealing at a greater magnificationthan the magnification of FIG. 40.

In some cases, the process that was used to apply a layer to the workingsubstrate may provide identifiable structural characteristic(s) to thatlayer that is/are distinct from structural characteristic(s) of otherlayers provided by other processes. More specifically, it may bepossible to identify how a layer was manufactured. For example, aportion of a substrate may be examined using a scanning electronmicroscope (SEM) to identify how one or more layers of the substratewere manufactured.

FIG. 42 shows a schematic view of an exemplary imaging device or system1000, which may also be referred to as a microfluorometer, for purposesof demonstrating functional arrangement for at least some opticalcomponents. The imaging device 1000 may detect light emissions (e.g.,fluorescent light emissions) from a structured substrate, such as thestructured substrates described herein. Two excitation sources areshown, including a green LED (LEDG) and a red LED (LEDR). Excitationlight from each passes through a green LED collector lens (L6) and redLED collector lens (L7), respectively. An LED fold mirror (M1) reflectsthe green excitation radiation to a combiner dichroic (F5) whichreflects the green excitation radiation through an excitation filter(F2), then through a laser diode beam splitter (F3), then through anexcitation field stop (FS), then through an excitation projection lensgroup L2 to an excitation/emission dichroic (F4) which reflects thegreen excitation radiation through a stationary objective lens group(L3) and a translating objective lens group (L4) to the surface of aflow cell (FC). The red excitation radiation passes from the red LEDcollector lens (L7) to the combiner dichroic (F5) after which the redexcitation radiation follows the same path as the green excitationradiation to the surface of the flow cell (FC). As shown in the figure,focusing is actuated by moving the translating objective lens group (L4)up and down (i.e. along the z dimension). Emission from the flow cell(FC) surface passes back through the translating objective lens group(L4), and then through the stationary objective lens group (L3) to theexcitation/emission dichroic (F4) which passes the emission radiation tothe emission projection les group (L1) through to the emission filterand then to the CMOS image sensor (Si). A laser diode (LD) is alsodirected via a laser diode coupling lens group (L5) to the laser diodebeam splitter (F3) which reflects the laser diode radiation through theexcitation field stop (FS), the excitation projection lens group (L2),the excitation/emission dichroic (F4), the stationary objective lensgroup (L3) and the translating objective lens group (L4) to the flowcell (FC).

As demonstrated by the exemplary embodiment of FIG. 42, the imagingdevice 1000 can include a beam splitter and a detector, wherein the beamsplitter is positioned to direct excitation radiation from an excitationradiation source to the objective lens and to direct emission radiationfrom the objective to the detector. The imaging device 1000 canoptionally include an excitation radiation source such as an LED.

It will be understood that the particular components shown in thefigures are exemplary and can be replaced with components of similarfunction. For example, any of a variety of radiation sources can be usedinstead of an LED. Particularly useful radiation sources are arc lamps,lasers, semiconductor light sources (SLSs), or laser diodes. LEDs can bepurchased, for example, from Luminus (Billerica, Mass.). Similarly, avariety of detectors are useful including, but not limited to acharge-coupled device (CCD) sensor; photomultiplier tubes (PMT's); orcomplementary metal-oxide-semiconductor (CMOS) sensor. A particularlyuseful detector is a 5-megapixel CMOS sensor (MT9P031) available fromAptina Imaging (San Jose, Calif).

FIG. 42 provides exemplary embodiments of an imaging device 1000 thatincludes two excitation sources. This configuration is useful fordetecting at least two fluorophores that are excited at differentwavelengths, respectively. If desired, the imaging device 1000 can beconfigured to include more than two excitation sources. For example, theimaging device 1000 can include at least 2, 3, 4 or more differentexcitation sources (i.e. sources producing different wavelengths fromeach other). Alternatively or additionally, beam splitters and opticalfilters can be used to expand the range of excitation wavelengthsavailable from an individual radiation source.

FIG. 43 shows an exemplary arrangement of four imaging devices (referredto as microfluorometers) in a single read head or carriage 1100. Thefour microfluorometers are arranged in a staggered layout with respectto first and second channels 1102 and 1104 of a flow cell 1106. In thearrangement shown, two of the microfluorometers (corresponding todetectors 1110A and 1110C) are configured to image separate regions ofthe first channel 1102 and the other two microfluorometers(corresponding to detectors 1110B and 1110D) are configured to imageseparate regions of the second channel 1104. As shown, themicrofluorometers (corresponding to detectors 1110A and 1110C) arestaggered with respect to the microfluorometers (corresponding todetectors 1110B and 1110D) in the x dimension such that the two pairs ofmicrofluorometers can detect the adjacent first and second channels 1102and 1104 respectively.

In the exemplary embodiment shown in FIG. 43 the four radiation sourcesare in thermal contact with a single large heat sink 1114. A singlelarge heat sink provides a greater degree of heat dissipation than manyconfigurations that use an individual heat sink for each radiationsource. However, if desired individual radiation sources can bethermally coupled to individual heat sinks. An advantage of thearrangement of microfluorometers shown in FIG. 43 is the provision of acompact read head. Similar advantages can be derived for embodimentswhere the relative positions of the excitation source and detector ineach microfluorometer are exchanged,

A microfluorometer, or read head having several microfluorometers, canbe positioned above a flow cell (with respect to gravity's arrow) asexemplified for several embodiments set forth herein. However, it isalso possible to position a microfluorometer, or a read head, underneatha flow cell. Accordingly a flow cell can be transparent on the top side,bottom side or both sides with respect to the wavelengths of excitationand emission radiation used. Indeed, in some embodiments it may bedesirable to position microfluorometers on both sides of a flow cell orto position read heads on both sides of a flow cell. Other orientationswith respect to gravity are also possible, including for example a sideto side orientation between a flow cell and microfluorometer (or readhead).

A microfluorometer or read head can be configured to detect the twoopposing, inner surfaces of a flow cell from a single side of the flowcell. For example, the microfluorometer or read head can employ anoptical compensator that is inserted and removed to detect alternativesurfaces of the flow cell. Exemplary methods and apparatus for detectingopposing inner surfaces of a flow cell such as the use of opticalcompensators are described in U.S. Pat. No. 8,039,817, which isincorporated herein by reference in its entirety. A compensator isoptional, for example, depending upon the NA and/or optical resolutionof the apparatus.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Solid supports can optionally be inert to a chemistry that is used tomodify a gel. For example, a solid support can be inert to chemistryused to attach analytes, such as nucleic acids, to gels in a method setforth herein. Exemplary solid supports include, but are not limited to,glass and modified or functionalized glass, plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

Particular embodiments of the methods and compositions presented hereinutilize a solid support having a patterned or structured substrate. Thepatterned or structured substrate can comprise a patterned gel array, asdescribed in U.S. Ser. No. 13/787,396, the entire content of which isincorporated herein by reference. In particular embodiments, astructured substrate can be made by patterning a solid support materialwith wells (e.g. microwells or nanowells), coating the patterned supportwith a gel material (e.g. PAZAM, SFA or chemically modified variantsthereof, such as the azidolyzed version of SFA (azido-SFA)) andpolishing the gel coated support, for example via chemical or mechanicalpolishing, thereby retaining gel in the wells but removing orinactivating substantially all of the gel from the interstitial regionson the surface of the structured substrate between the wells. Primernucleic acids can be attached to gel material. A solution of targetnucleic acids (e.g. a fragmented human genome) can then be contactedwith the polished substrate such that individual target nucleic acidswill seed individual wells via interactions with primers attached to thegel material; however, the target nucleic acids will not occupy theinterstitial regions due to absence or inactivity of the gel material.Amplification of the target nucleic acids will be confined to the wellssince absence or inactivity of gel in the interstitial regions preventsoutward migration of the growing nucleic acid colony. The process isconveniently manufacturable, being scalable and utilizing conventionalmicro- or nano-fabrication methods.

A solid support used in a structured substrate set forth herein can bemade from any of a variety of materials set forth herein, for example,above in the definitions, below in the examples or immediatelyfollowing. A particularly useful material is glass. Other suitablesubstrate materials may include polymeric materials, plastics, silicon,quartz (fused silica), borofloat glass, silica, silica-based materials,carbon, metals, an optical fiber or optical fiber bundles, sapphire, orplastic materials such as COCs and epoxies. The particular material canbe selected based on properties desired for a particular use. Forexample, materials that are transparent to a desired wavelength ofradiation are useful for analytical techniques that will utilizeradiation of the desired wavelength, such as one or more of thetechniques set forth herein. Conversely, it may be desirable to select amaterial that does not pass radiation of a certain wavelength (e.g.being opaque, absorptive, or reflective). This can be useful forformation of a mask to be used during manufacture of the structuredsubstrate, such as a method set forth herein; or to be used for achemical reaction or analytical detection carried out using thestructured substrate, such as those set forth herein. Other propertiesof a material that can be exploited are inertness or reactivity tocertain reagents used in a downstream process, such as those set forthherein; or ease of manipulation or low cost during a manufacturingprocess manufacture, such as those set forth herein. Further examples ofmaterials that can be used in the structured substrates or methods ofthe present disclosure are described in U.S. Ser. No. 13/661,524 and USPat. App. Pub. No. 2012/0316086 A1, each of which is incorporated hereinby reference.

Particularly useful solid supports for some embodiments are locatedwithin a flow cell apparatus. Exemplary flow cells, methods for theirmanufacture and methods for their use are described in US Pat. App.Publ. Nos. 2010/0111768 A1 or 2012-0270305 A1; or WO 05/065814, each ofwhich is incorporated herein by reference. Flow cells provide aconvenient format for housing an array that is produced by the methodsof the present disclosure and that is subjected to asequencing-by-synthesis (SBS) or other technique that involves repeateddelivery of reagents in cycles (e.g. synthesis techniques or detectiontechniques having repetitive or cyclic steps). Exemplary detectionmethods are set forth in further detail below.

In some embodiments a flow-cell or other vessel having multiple surfacesis used. Vessels having multiple surfaces can be used such that only asingle surface has gel-containing concave features (e.g. wells).Alternatively two or more surfaces present in the vessel can havegel-containing concave features. One or more surfaces of a flow cell canbe selectively detected. For example, opposing surfaces in the interiorof a flow cell can be selectively addressed with focused radiation usingmethods known in the art such as confocal techniques. Useful confocaltechniques and devices for selectively directing radiation to multiplesurfaces of a vessel (e.g. a flow cell) are described, for example, inUS Pat. App. Pub. No. 2009/0272914 A1 or U.S. Pat. No. 8,039,817, eachof which is incorporated herein by reference.

In many embodiments, the interstitial region can be substantially devoidof nanostructures by polishing the solid support, for example viachemical or mechanical polishing, thereby retaining nanostructures inthe wells but removing or inactivating substantially all of thenanostructures from the interstitial regions on the surface of thestructured substrate between the wells. Mechanical polishing can becarried out by applying abrasive forces to the surface of the solidsupport. Exemplary methods include abrasion with a slurry of beads,wiping with a sheet or cloth, scraping or the like. It will beunderstood that beads used for polishing or other uses set forth hereincan be, but need not be, spherical. Rather beads can have irregularshapes, polygonal shapes, ovoid shapes, elongated shapes, cylindricalshapes etc. The surface of the beads can be smooth or rough. Any of avariety of particles can be useful as beads for the methods andcompositions set forth herein. One example of polishing includes using alintless (cleanroom grade) wipe coated with a 3 μm silica bead slurry(10% w/v in water) to remove interstitial nanostructures. A polishingwheel/grinder can also be used with this slurry. Mechanical polishingcan also be achieved using a fluid jet or gas (e.g. air or inert gassuch as Argon or Nitrogen) jet to remove gel from interstitial regions.

As used herein, the term “library,” when used in reference to analytes,refers to a collection of analytes having different chemicalcompositions. Typically, the analytes in a library will be differentspecies having a common feature or characteristic of a genera or class,but otherwise differing in some way. For example, a library can includenucleic acid species that differ in nucleotide sequence, but that aresimilar with respect to having a sugar-phosphate backbone.

As used herein, the terms “nucleic acid” and “nucleotide” are intendedto be consistent with their use in the art and to include naturallyoccurring species or functional analogs thereof. Particularly usefulfunctional analogs of nucleic acids are capable of hybridizing to anucleic acid in a sequence specific fashion or capable of being used asa template for replication of a particular nucleotide sequence.Naturally occurring nucleic acids generally have a backbone containingphosphodiester bonds. An analog structure can have an alternate backbonelinkage including any of a variety of those known in the art. Naturallyoccurring nucleic acids generally have a deoxyribose sugar (e.g. foundin deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found inribonucleic acid (RNA)). A nucleic acid can contain nucleotides havingany of a variety of analogs of these sugar moieties that are known inthe art. A nucleic acid can include native or non-native nucleotides. Inthis regard, a native deoxyribonucleic acid can have one or more basesselected from the group consisting of adenine, thymine, cytosine orguanine and a ribonucleic acid can have one or more bases selected fromthe group consisting of uracil, adenine, cytosine or guanine. Usefulnon-native bases that can be included in a nucleic acid or nucleotideare known in the art. The terms “probe” or “target,” when used inreference to a nucleic acid, are intended as semantic identifiers forthe nucleic acid in the context of a method or composition set forthherein and does not necessarily limit the structure or function of thenucleic acid beyond what is otherwise explicitly indicated. The terms“probe” and “target” can be similarly applied to other analytes such asproteins, small molecules, cells or the like.

As used herein, the terms “coat” and “coating” and like terms, when usedas a verb, are intended to mean providing a layer or covering on asurface. At least a portion of the surface can be provided with a layeror cover. In some cases the entire surface can be provided with a layeror cover. In alternative cases only a portion of the surface will beprovided with a layer or covering. The term “coat,” when used todescribe the relationship between a surface and a material, is intendedto mean that the material is present as a layer or cover on the surface.The material can seal the surface, for example, preventing contact ofliquid or gas with the surface. However, the material need not form aseal. For example, the material can be porous to liquid, gas, or one ormore components carried in a liquid or gas. Exemplary materials that cancoat a surface include, but are not limited to, a gel, polymer, organicpolymer, liquid, metal, a second surface, plastic, silica, or gas.

Structured substrates of the present disclosure that contain nucleicacid arrays can be used for any of a variety of purposes. A particularlydesirable use for the nucleic acids is to serve as capture probes thathybridize to target nucleic acids having complementary sequences. Thetarget nucleic acids once hybridized to the capture probes can bedetected, for example, via a label recruited to the capture probe.Methods for detection of target nucleic acids via hybridization tocapture probes are known in the art and include, for example, thosedescribed in U.S. Pat. Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431or US Pat. App. Pub. Nos. 2005/0053980 A1; 2009/0186349 A1 or2005/0181440 A1, each of which is incorporated herein by reference. Forexample, a label can be recruited to a capture probe by virtue ofhybridization of the capture probe to a target probe that bears thelabel. In another example, a label can be recruited to a capture probeby hybridizing a target probe to the capture probe such that the captureprobe can be extended by ligation to a labeled oligonucleotide (e.g. vialigase activity) or by addition of a labeled nucleotide (e.g. viapolymerase activity).

A nucleic acid array can also be used in a sequencing procedure, such asa sequencing-by-synthesis (SBS) technique. Briefly, SBS can be initiatedby contacting the target nucleic acids with one or more labelednucleotides, DNA polymerase, etc. Those features where a primer isextended using the target nucleic acid as template will incorporate alabeled nucleotide that can be detected. Optionally, the labelednucleotides can further include a reversible termination property thatterminates further primer extension once a nucleotide has been added toa primer. For example, a nucleotide analog having a reversibleterminator moiety can be added to a primer such that subsequentextension cannot occur until a deblocking agent is delivered to removethe moiety. Thus, for embodiments that use reversible termination, adeblocking reagent can be delivered to the flow cell (before or afterdetection occurs). Washes can be carried out between the variousdelivery steps. The cycle can then be repeated n times to extend theprimer by n nucleotides, thereby detecting a sequence of length n.Exemplary SBS procedures, fluidic systems and detection platforms thatcan be readily adapted for use with an array produced by the methods ofthe present disclosure are described, for example, in Bentley et al.,Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO 07/123744; U.S.Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, andUS Pat. App. Pub. No. 2008/0108082 A1, each of which is incorporatedherein by reference.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and 6,274,320, each of which is incorporated herein by reference). Inpyrosequencing, released PPi can be detected by being converted toadenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATPcan be detected via luciferase-produced photons. Thus, the sequencingreaction can be monitored via a luminescence detection system.Excitation radiation sources used for fluorescence based detectionsystems are not necessary for pyrosequencing procedures. Useful fluidicsystems, detectors and procedures that can be used for application ofpyrosequencing to arrays of the present disclosure are described, forexample, in WIPO Pat. App. Ser. No. PCT/US11/57111, US Pat. App. Pub.No. 2005/0191698 A1, U.S. Pat. No. 7,595,883, and U.S. Pat. No.7,244,559, each of which is incorporated herein by reference.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. No. 5,599,675; and U.S. Pat. No. 5,750,341, each of which isincorporated herein by reference. Some embodiments can includesequencing-by-hybridization procedures as described, for example, inBains et al., Journal of Theoretical Biology 135(3), 303-7 (1988);Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al.,Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which isincorporated herein by reference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, nucleic acids that are presentin gel-containing wells (or other concave features) are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein, or in references citedherein, can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides. Techniquesand reagents for FRET-based sequencing are described, for example, inLevene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett.33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105,1176-1181 (2008), the disclosures of which are incorporated herein byreference.

Another useful application for an array of the present disclosure isgene expression analysis. Gene expression can be detected or quantifiedusing RNA sequencing techniques, such as those, referred to as digitalRNA sequencing. RNA sequencing techniques can be carried out usingsequencing methodologies known in the art such as those set forth above.Gene expression can also be detected or quantified using hybridizationtechniques carried out by direct hybridization to an array or using amultiplex assay, the products of which are detected on an array. Anarray of the present disclosure can also be used to determine genotypesfor a genomic DNA sample from one or more individual. Exemplary methodsfor array-based expression and genotyping analysis that can be carriedout on an array of the present disclosure are described in U.S. Pat.Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US Pat. App. Pub.Nos. 2005/0053980 A1; 2009/0186349 A1 or 2005/0181440 A1, each of whichis incorporated herein by reference.

Several applications for arrays of the present disclosure have beenexemplified above in the context of ensemble detection, wherein multiplecopies of a target nucleic acid are present at each feature and aredetected together. In alternative embodiments, a single nucleic acid,whether a target nucleic acid or amplicon thereof, can be detected ateach feature. For example, a gel-containing well (or other concavefeature) can be configured to contain a single nucleic acid moleculehaving a target nucleotide sequence that is to be detected. Any of avariety of single molecule detection techniques can be used including,for example, modifications of the ensemble detection techniques setforth above to detect the sites at increased resolution or using moresensitive labels. Other examples of single molecule detection methodsthat can be used are set forth in US Pat. App. Pub. No. 2011/0312529 A1;U.S. Ser. No. 61/578,684; and U.S. Ser. No. 61/540,714, each of which isincorporated herein by reference.

As used herein, the term “well” refers to a discrete concave feature ina solid support having a surface opening that is completely surroundedby interstitial region(s) of the surface. Wells can have any of avariety of shapes at their opening in a surface including but notlimited to round, elliptical, square, polygonal, star shaped (with anynumber of vertices) etc. The cross section of a well taken orthogonallywith the surface can be curved, square, polygonal, hyperbolic, conical,angular, etc.

As used herein, the term “concave feature,” when used in reference to asolid support, refers to a recess or indentation in the solid support.Exemplary concave features include, but are not limited to, a well, pit,hole, depression, channel, or trough. A concave feature can optionallyhave a curved cross section (in the dimension orthogonal to the surfaceof the solid support); however, a cross section with one or more linearsections, angles or corners is also possible. Cross sections withcombinations of curved and linear sections are also possible. Generally,a concave feature need not pass completely through the solid support,for example, instead having a bottom surface or point in the substrate.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

For example, in an embodiment, a structured substrate is provided thatincludes a substrate body having an active side. The substrate bodyincludes reaction cavities that open along the active side andinterstitial regions that separate the reaction cavities. The structuredsubstrate also includes an ensemble amplifier positioned within each ofthe reaction cavities. The ensemble amplifier includes a plurality ofnanostructures that are configured to at least one of amplifyelectromagnetic energy that propagates into the corresponding reactioncavity or amplify electromagnetic energy that is generated within thecorresponding reaction cavity.

In one or more aspects, the nanostructures for each of the ensembleamplifiers may have a predetermined position relative to the othernanostructures of the corresponding ensemble amplifier. The ensembleamplifiers may have essentially the same arrangement of nanostructures.Optionally, the ensemble amplifiers have a polarized configuration suchthat a response from the ensemble amplifiers is based on a polarizationof the electromagnetic energy, wherein adjacent ensemble amplifiers havedifferent polarized configurations.

In one or more aspects, the reaction cavities may include a first set ofreaction cavities and a second set of reaction cavities. The first setof reaction cavities may preferentially respond to a first polarizedlight over a second polarized light and the second set of reactioncavities may preferentially respond to the second polarized light overthe first polarized light.

In one or more aspects, the active side may include a side surface thatextends along the interstitial regions. The side surface may besubstantially planar.

In one or more aspects, an organic material may be disposed within thereaction cavities and cover the nanostructures. The organic material maybe configured to immobilize a biomolecule within the correspondingreaction cavity. Optionally, the organic material comprises a gelmaterial. Optionally, the organic material comprises a hydrogel.Optionally, the organic material has a volume that is configured toaccommodate only a single analyte such that steric exclusion preventsmore than one analyte from being captured or seeding the reactioncavity. Optionally, the organic material is permeable to liquid and isconfigured to attach to a nucleic acid.

In one or more aspects, the substrate body may include a base layerhaving the nanostructures projecting therefrom. The substrate body mayalso include a cavity layer stacked with respect to the base layer. Thecavity layer may be shaped to include the reaction cavities. Optionally,the nanostructures extend from the base layer, through a portion of thecavity layer, and into the corresponding reaction cavities.

In one or more aspects, the nanostructures are formed of a plasmonresonant material.

In one or more aspects, the nanostructures comprises at least one of:Gold (Au), Silver (Ag), Tin (Sn) Rhodium (Rh), Ruthenium (Ru), Palladium(Pd), Osmium (Os), Iridium (Ir), Platinum (Pt), Titanium (Ti), Aluminum(Al), Chromium (Cr), Copper (Cu), p-type doped silicon, n-type dopedsilicon, gallium arsenide, Zinc-Indium-Tin Oxide (ZITO), or TantalumOxide.

In one or more aspects, the nanostructures in the ensemble amplifiersmay have a material composition, shape, and relative position withrespect to other nanostructures of the ensemble amplifier to at leastone of amplify the electromagnetic energy that propagates into thecorresponding reaction cavity or amplify the electromagnetic energy thatis generated within the corresponding reaction cavity.

In one or more aspects, the nanostructures in the ensemble amplifiershave a material composition, shape, and relative position with respectto other nanostructures of the ensemble amplifier to amplify theelectromagnetic energy that is generated within the correspondingreaction cavity. Optionally, the electromagnetic energy includesfluorescent light emissions.

In one or more aspects, the nanostructures in the ensemble amplifiersmay have a composition, shape, and relative position with respect toother nanostructures of the ensemble amplifier to amplify theelectromagnetic energy that propagates into the corresponding reactioncavity.

In one or more aspects, a wavelength of the excitation light or thelight emissions is between 300 nanometers (nm) and 750 nm.

In one or more aspects, each of the nanostructures may include ananobody comprising a nanoimprint-lithography (NIL) material and anexternal layer that surrounds the nanobody. Optionally, the externallayer comprises at least one of: Gold (Au), Silver (Ag), Tin (Sn)Rhodium (Rh), Ruthenium (Ru), Palladium (Pd), Osmium (Os), Iridium (Ir),Platinum (Pt), Titanium (Ti), Aluminum (Al), Chromium (Cr), Copper (Cu),p-type doped silicon, n-type doped silicon, gallium arsenide,Zinc-Indium-Tin Oxide (ZITO), or Tantalum Oxide.

In one or more aspects, a passivation layer may extend over thenanobodies. In one or more aspects, a device cover may be coupled to thesubstrate body to form a flow channel between the active side of thesubstrate body and the device cover. The flow channel is configured todirect a flow of liquid therethrough that flows into the reactioncavities.

In one or more aspects, the reaction cavities have corresponding bottomsurfaces. The nanostructures may project from the bottom surface of thecorresponding reaction cavity toward the active side.

In one or more aspects, each of the reaction cavities may be defined byat least one sidewall that extends between the active side and a bottomsurface of the reaction cavity. The nanostructures form at least aportion of the at least one sidewall. Optionally, the nanostructuresproject from the bottom surface of the corresponding reaction cavity.

In one or more aspects, the interstitial regions may be substantiallydevoid of the nanostructures. Alternatively, the interstitial regionsmay have embedded nanostructures.

In one or more aspects, the nanostructures may have a height thatextends toward the active side along an elevation axis. The height maybe at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90nm or 100 nm.

In one or more aspects, the nanostructures may have a height thatextends toward the active side along an elevation axis. Thenanostructures may have a cross-sectional dimension taken transverse tothe elevation axis. The cross-sectional dimension may be at least 10 nm,20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm.

In one or more aspects, the nanostructures may have a height thatextends toward the active side along an elevation axis. Thenanostructures may have a cross-sectional dimension taken transverse tothe elevation axis. The cross-sectional dimension may be less than 100nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm.Optionally, the cross-sectional dimension is a diameter. Optionally, thecross-sectional dimension represents the greatest cross-sectionaldimension that can be taken through the nanostructure.

In one or more aspects, the nanostructures may include dimers or trimerswithin the reaction cavities.

In one or more aspects, the ensemble amplifiers may form bowtienanoantennas. In one or more aspects, the nanostructures comprisenanorods, nanorings, and/or nanoplugs.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method may include providing a base layer having a baseside and forming nanostructures along the base side of the base layer.The method may also include forming a cavity layer that is stacked abovethe base side. The cavity layer includes a plurality of reactioncavities in which each reaction cavity includes a plurality of thenanostructures therein. The plurality of nanostructures form an ensembleamplifier of the corresponding reaction cavity that is configured to atleast one of amplify electromagnetic energy propagating into thecorresponding reaction cavity or amplify electromagnetic energygenerated within the corresponding reaction cavity.

Various features of the structured substrate, the nanostructures, and/orthe ensemble amplifiers may be similar to those described herein.

In one or more aspects, the method also includes providing an organicmaterial within the reaction cavities such that the organic materialcovers the nanostructures. The organic material may be configured toimmobilize a biomolecule within the corresponding reaction cavity.

Optionally, the method also includes polishing the active side to removethe organic material from interstitial regions.

In one or more aspects, the method also includes mounting a device coverto the substrate body to form a flow channel between the active side ofthe substrate body and the device cover. The flow channel may beconfigured to direct a flow of liquid therethrough that flows into thereaction cavities.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base sideand forming nanostructures along the base side of the base layer. Themethod also includes providing a nanoimprint lithography (NIL) layerover the array of nanostructures and imprinting an array of reactioncavities into the NIL layer. A different sub-array of the nanostructuresis positioned under each reaction cavity. Each sub-array ofnanostructures may be surrounded by a respective fill region of the NILlayer. The method also includes removing the respective fill regions ofthe NIL layer to expose the sub-arrays of nanostructures within thecorresponding reactions cavities. The sub-array of nanostructures withineach reaction cavity forms an ensemble amplifier of the correspondingreaction cavity that is configured to at least one of amplifyelectromagnetic energy propagating into the corresponding reactioncavity or amplify electromagnetic energy generated within thecorresponding reaction cavity.

Various features of the structured substrate, the nanostructures, and/orthe ensemble amplifiers may be similar to those described herein.

For example, in one or more aspects, the NIL layer is a top NIL layer,wherein forming the nanostructures includes providing a bottom NIL layerand imprinting the nanostructures.

Various features of the structured substrate, the nanostructures, and/orthe ensemble amplifiers may be similar to those described herein.

In one or more aspects, the method also includes providing an organicmaterial within the reaction cavities such that the organic materialcovers the nanostructures. The organic material may be configured toimmobilize a biomolecule within the corresponding reaction cavity.

In one or more aspects, the method also includes polishing the activeside to remove the organic material from interstitial regions.

In one or more aspects, the method also includes mounting a device coverto the substrate body to form a flow channel between the active side ofthe substrate body and the device cover, the flow channel configured todirect a flow of liquid therethrough that flows into the reactioncavities.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base sideand providing a nanoimprint lithography (NIL) layer along the base side.The method also includes imprinting the NIL layer to form a base portionand an array of nanobodies that project from the base portion. Themethod also includes depositing a plasmon resonant film that covers thenanobodies to form a plurality of nanostructures. Each nanostructureincludes a corresponding nanobody and a portion of the plasmon resonantfilm. The method also includes forming a cavity layer having a pluralityof reaction cavities in which each reaction cavity includes a pluralityof the nanostructures therein. The plurality of nanostructures form anensemble amplifier of the corresponding reaction cavity that isconfigured to at least one of amplify electromagnetic energy propagatinginto the corresponding reaction cavity or amplify electromagnetic energygenerated within the corresponding reaction cavity.

Various features of the structured substrate, the nanostructures, and/orthe ensemble amplifiers may be similar to those described herein.

For example, the cavity layer may include a NIL material and wherein thestep or operation of forming the cavity layer may include imprinting theNIL material of the cavity layer to form the reaction cavities.

In one or more aspects, the method also includes providing an organicmaterial within the reaction cavities such that the organic materialcovers the nanostructures. The organic material may be configured toimmobilize a biomolecule within the corresponding reaction cavity.

In one or more aspects, the method may also include polishing the activeside to remove the organic material from interstitial regions.

In one or more aspects, the method may also include mounting a devicecover to the substrate body to form a flow channel between the activeside of the substrate body and the device cover. The flow channel may beconfigured to direct a flow of liquid therethrough that flows into thereaction cavities.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having aside surface and an array of reaction cavities. Each of the reactioncavities has an opening along the side surface and extends a depth fromthe corresponding opening into the working substrate. The reactioncavities coincide with an array plane. The method may also includedirecting a deposition stream onto the working substrate at anon-orthogonal angle with respect to the array plane. The depositionstream includes a plasmon resonant material. The working substrate formsa shadow area and an incident area in each reaction cavity relative to apath of the deposition stream such that the plasmon resonant material ofthe deposition stream is blocked by the side surface from beingdeposited onto the shadow area and is permitted to pass through theopening and form along the incident area.

In one or more aspects, the reaction cavities are defined by respectiveside walls and bottom surfaces. The side walls extend away from the sidesurface toward the respective bottom surface. The incident area mayextend along at least a portion of the side wall. The shadow area mayextend along at least a portion of the bottom surface.

In one or more aspects, the method also includes forming a structuredsubstrate for analyzing biomolecules that includes the workingsubstrate. The material may be deposited along the incident areasforming at least portions of nanostructures that amplify electromagneticenergy.

In one or more aspects, the non-orthogonal angle is a firstnon-orthogonal angle, the shadow area is a first shadow area, thedeposition stream is a first deposition stream, and the incident area isa first incident area. The method may also include directing a seconddeposition stream onto the working substrate at a second non-orthogonalangle with respect to the array plane that is different than the firstnon-orthogonal angle. The working substrate may form a second shadowarea and a second incident area in each reaction cavity relative to thepath of the deposition stream such that a plasmon resonant material ofthe second deposition stream is blocked by the side surface from beingdeposited onto the second shadow area and is permitted to pass throughthe opening and form along the second incident area.

Optionally, at least a portion of the second incident area overlaps withthe first shadow area.

Optionally, the plasmon resonant material of the first and seconddepositions streams is the same.

Optionally, the plasmon resonant material of the first and seconddepositions streams is different.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes (a) providing a working substrate having aside surface and an array of reaction cavities. Each of the reactioncavities has an opening along the side surface and extends a depth fromthe corresponding opening into the working substrate. The reactioncavities coincide with an array plane. The method also includes (b)positioning the working substrate in a receiving orientation relative toa material source (c) directing a deposition stream from the materialsource onto the working substrate at a non-orthogonal angle with respectto the array plane. The deposition stream includes a plasmon resonantmaterial, wherein the working substrate forms a shadow area and anincident area in each reaction cavity when in the receiving orientationsuch that the plasmon resonant material from the deposition stream isblocked by the side surface from being deposited onto the shadow areaand is permitted to pass through the opening and form along the incidentarea.

In one or more aspects, the method includes repeating steps (a)-(c), forat least one series, at a different receiving orientation.

In one or more aspects, the method includes repeating steps (a)-(c), forat least one series, with a different plasmon resonant material.

Optionally, steps (a)-(c) are repeated to form an ensemble amplifierhaving a plurality of the nanostructures within each of the receivingcavities.

In an embodiment, a method of analyzing biomolecules capable ofgenerating light emissions is provided. The method may include providinga structured substrate having an array of reaction sites. Each of thereaction sites includes a plurality of nanostructures that form anensemble amplifier that is configured to amplify electromagnetic energythat is incident with the nanostructures of the ensemble amplifier. Thearray of reaction sites includes a first sub-array of reaction sites anda second sub-array of reaction sites. The ensemble amplifiers of thefirst sub-array are configured to preferentially respond to a firstpolarized excitation light. The ensemble amplifiers of the secondsub-array are configured to preferentially respond to a second polarizedexcitation light. The method also includes illuminating the array ofreaction sites with the first polarized excitation light and detectinglight emissions from the first sub-array. The method also includesilluminating the array of reaction sites with the second polarizedexcitation light and detecting light emissions from the secondsub-array.

In one or more aspects, the structured substrate includes reactioncavities that form the reaction sites. The reaction cavities extend adepth into the structured substrate. Each of the reaction cavities hasthe corresponding ensemble amplifier therein.

In one or more aspects, the ensemble amplifiers of the first sub-arrayhave a dipole moment that is essentially parallel to a polarization ofthe first polarized excitation light and the ensemble amplifiers of thesecond sub-array have a dipole moment that is essentially parallel to apolarization of the second polarized excitation light.

In one or more aspects, the light emissions include fluorescence.

In one or more aspects, the reaction sites are covered by a gel materialthat is configured to hold biomolecules.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a base layer having a base side,providing a feature layer along the base side, and forming nano-bodiesfrom the feature layer through reactive-ion etching (RIE). The methodalso includes coating the nano-bodies with a plasmon resonant materialand providing a passivation layer over the nano-bodies and the plasmonresonant material.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having aplurality of cavities. The method also includes providing a featurelayer that includes nano-bodies along the working substrate. The featurelayer fills the cavities. The method also includes removing materialwithin the cavities through reactive-ion etching (ME) to reveal thenano-bodies and providing a passivation layer over the nano-bodies.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having aplurality of cavities, directly depositing a deposition stream onto thebase layer a non-orthogonal angle, and transforming the deposited layerinto nano-bodies. The method also includes removing the deposited layerfrom interstitial regions and providing a passivation layer over thenano-bodies.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having aplurality of cavities, depositing a nanoimprint lithography (NIL)material that includes nano-bodies along the working substrate, andimprinting the NIL material to form a non-planar feature layer. Themethod also includes selectively removing the material to formnano-bodies and providing a passivation layer.

In an embodiment, a method of manufacturing a structured substrate isprovided. The method includes providing a working substrate having abase layer and forming an array of nano-bodies along the base layer. Themethod also includes depositing a plasmon resonant material along thenano-bodies and thermally annealing the plasmon resonant material toform nanoparticles along the nano-bodies. The method also includesproviding a passivation layer over the nanoparticles.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication.

As used herein, the terms “comprising,” “including,” and “having,” andthe like are intended to be open-ended, including not only the recitedelements, but possibly encompassing additional elements.

It is to be understood that the above description is intended to beillustrative, and not restrictive. For example, the above-describedembodiments (and/or aspects thereof) may be used in combination witheach other. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventivesubject matter without departing from its scope. Dimensions, types ofmaterials, orientations of the various components, and the number andpositions of the various components described herein are intended todefine parameters of certain embodiments, and are by no means limitingand are merely exemplary embodiments. Many other embodiments andmodifications within the spirit and scope of the claims will be apparentto those of skill in the art upon reviewing the above description. Thescope of the inventive subject matter should, therefore, be determinedwith reference to the appended claims, along with the full scope ofequivalents to which such claims are entitled.

As used in the description, the phrases “in an exemplary embodiment,”“in some embodiments,” “in particular embodiments,” and the like meansthat the described embodiment(s) are examples of embodiments that may beformed or executed in accordance with the present application. Thephrase is not intended to limit the inventive subject matter to thatembodiment. More specifically, other embodiments of the inventivesubject matter may not include the recited feature or structuredescribed with a particular embodiment.

In the appended claims, the terms “including” and “in which” are used asthe plain-English equivalents of the respective terms “comprising” and“wherein.” Moreover, in the following claims, the terms “first,”“second,” and “third,” etc. are used merely as labels, and are notintended to impose numerical requirements on their objects. Further, thelimitations of the following claims are not written inmeans—plus-function format and are not intended to be interpreted basedon 35 U.S.C. § 112 (f) unless and until such claim limitations expresslyuse the phrase “means for” followed by a statement of function void offurther structure.

The following claims recite one or more embodiments of the presentapplication and are hereby incorporated into the description of thepresent application.

What is claimed is:
 1. A method comprising: providing a structuredsubstrate having an array of reaction sites, each of the reaction sitesincluding a plurality of nanostructures that form an ensemble amplifierthat amplifies electromagnetic energy that is incident with thenanostructures of the ensemble amplifier, wherein the array of reactionsites include a first sub-array of reaction sites and a second sub-arrayof reaction sites, the ensemble amplifiers of the first sub-arrayconfigured to preferentially respond to a first polarized excitationlight, the ensemble amplifiers of the second sub-array configured topreferentially respond to a second polarized excitation light;illuminating the array of reaction sites with the first polarizedexcitation light; detecting light emissions from the first sub-array;illuminating the array of reaction sites with the second polarizedexcitation light; and detecting light emissions from the secondsub-array.
 2. The method of claim 1, wherein the structured substrateincludes reaction cavities that form the reaction sites, the reactioncavities extending a depth into the structured substrate, each of thereaction cavities having the ensemble amplifier of the reaction sitetherein.
 3. The method of claim 1, wherein the ensemble amplifiers ofthe first sub-array have a dipole moment that is essentially parallel toa polarization of the first polarized excitation light and the ensembleamplifiers of the second sub-array have a dipole moment that isessentially parallel to a polarization of the second polarizedexcitation light.
 4. The method of claim 1, wherein the structuredsubstrate includes reaction cavities that form the reaction sites, thereaction cavities extending a depth into the structured substrate, eachof the reaction cavities having the ensemble amplifier of the reactionsite therein; and wherein the ensemble amplifiers of the first sub-arrayhave a dipole moment that is essentially parallel to a polarization ofthe first polarized excitation light and the ensemble amplifiers of thesecond sub-array have a dipole moment that is essentially parallel to apolarization of the second polarized excitation light.
 5. The method ofclaim 1, wherein the light emissions include fluorescence.
 6. The methodof claim 1, wherein the reaction sites are covered by a gel materialthat is configured to hold biomolecules.
 7. The method of claim 1,wherein an organic material covers the nanostructures, where the organicmaterial immobilizes a biomolecule within the corresponding reactionsite.
 8. The method of claim 4, wherein an organic material covers thenanostructures, the organic material configured to immobilize abiomolecule within the corresponding reaction cavity.
 9. A methodcomprising: providing a base layer having a base side; providing ananoimprint lithography (NIL) layer along the base side; imprinting theNIL layer to form a base portion and an array of nanobodies that projectfrom the base portion; depositing a plasmon resonant film that coversthe nanobodies to form a plurality of nanostructures, each nanostructureincluding a corresponding nanobody and a portion of the plasmon resonantfilm; and forming a cavity layer including a plurality of reactioncavities in which each reaction cavity includes a plurality of thenanostructures therein, the plurality of nanostructures forming anensemble amplifier of the corresponding reaction cavity that isconfigured to at least one of amplify electromagnetic energy propagatinginto the corresponding reaction cavity or amplify electromagnetic energygenerated within the corresponding reaction cavity.
 10. The method ofclaim 9, wherein the cavity layer comprises a NIL material and whereinforming the cavity layer includes imprinting the NIL material of thecavity layer to form the reaction cavities.
 11. The method of claim 9,wherein the nanostructures for each of the ensemble amplifiers have apredetermined position relative to the other nanostructures of thecorresponding ensemble amplifier, wherein the ensemble amplifiers haveessentially the same arrangement of nanostructures.
 12. The method ofclaim 9, wherein the ensemble amplifiers have a polarized configurationsuch that a response from the ensemble amplifiers is based on apolarization of the electromagnetic energy, where adjacent ensembleamplifiers have different polarized configurations.
 13. The method ofclaim 9, wherein the reaction cavities include a first set of reactioncavities and a second set of reaction cavities, the first set ofreaction cavities preferentially responding to a first polarized lightover a second polarized light and the second set of reaction cavitiespreferentially responding to the second polarized light over the firstpolarized light.
 14. The method of claim 9, providing an organicmaterial within the reaction cavities such that the organic materialcovers the nanostructures, the organic material configured to immobilizea biomolecule within the corresponding reaction cavity.
 15. The methodof claim 14, wherein the organic material comprises a hydrogel.
 16. Themethod of claim 14, further comprising polishing the active side toremove the organic material from interstitial regions.
 17. The method ofclaim 14, wherein the organic material has a volume that is configuredto accommodate only a single analyte such that steric exclusion preventsmore than one analyte from being captured or seeding the reactioncavity.
 18. The method of claim 9, wherein the nanostructures in theensemble amplifiers have a material composition, shape, and relativeposition with respect to other nanostructures of the ensemble amplifierto at least one of amplify the electromagnetic energy that propagatesinto the corresponding reaction cavity or amplify the electromagneticenergy that is generated within the corresponding reaction cavity. 19.The method of claim 9, wherein the nanostructures in the ensembleamplifiers have a material composition, shape, and relative positionwith respect to other nanostructures of the ensemble amplifier toamplify the electromagnetic energy that is generated within thecorresponding reaction cavity.
 20. The method of claim 9, furthercomprising mounting a device cover to the substrate body to form a flowchannel between the active side of the substrate body and the devicecover, the flow channel configured to direct a flow of liquidtherethrough that flows into the reaction cavities. 6